WO2013075233A1 - Method for treating brain cancer - Google Patents

Abstract

The present invention relates to novel compositions and therapeutic methods for the treatment of cancer, in particular malignant glioma, breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma. The compositions include RNAi molecules, antisense RNAs or vectors encoding them which reduce expression of downregulated in renal cell carcinoma (DRR) in tumor cells, and inhibit tumor cell invasion, e.g., malignant glioma cell invasion.

Description

METHOD FOR TREATING BRAIN CANCER

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.

61 /562,098 filed November 21 , 201 1 , the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel pharmaceutical compositions and methods for treating malignant glioma and other invasive cancers such as breast carcinoma, prostate carcinoma, and squamous cell carcinoma.

BACKGROUND OF THE INVENTION

Gliomas arise from the supporting cells of the brain, called the glia.

These cells are subdivided into astrocytes, ependymal cells and

oligodendroglial cells. Gliomas are the most common primary brain cancers and are amongst the most devastating of human malignancies. The tumors are graded from the lowest grade 1 to highest grade 4, with glioblastoma multiforme (GBM) being the highest grade and deadliest type of glioma. High-grade glioma or GBM is the most common primary malignant brain tumor, as well as the most devastating, accounting for 19 percent of all primary brain tumors.

Benign gliomas, known as pilocytic astrocytomas, are seen in children and are very well treated by complete surgical resection, with patients typically maintaining a full life expectancy. In contrast, high-grade or malignant gliomas, known as astrocytomas, oligodendrogliomas or glioblastomas, are adult neoplasms characterized by brain invasion. Unlike benign gliomas which do not invade normal brain, malignant gliomas are highly invasive. As a rule, high-grade gliomas almost always grow back even after complete surgical excision.

Malignant gliomas can be further divided into low grade and high grade. Low grade malignant gliomas are highly invasive but have low proliferation rates, often invading multiple lobes prior to clinical presentation. Over time, low grade malignant gliomas may incur genetic changes that increase their proliferation rate and convert them to a higher grade (Louis, D.N. et al., Cancer Cell 1 : 125-128, 2002).

The prognosis for patients with high-grade gliomas is generally poor.

Malignant gliomas are among the most challenging of all cancers to treat successfully because they are characterized not only by aggressive proliferation and expansion, but also by their aggressive invasion of distant brain tissue. Of approximately 10,000 Americans diagnosed each year with malignant gliomas, about half are alive 1 year after diagnosis, and 25% after two years. Those with anaplastic astrocytoma survive about three years. Glioblastoma multiforme has a worse prognosis with less than 12 month survival after diagnosis. Standard treatment includes surgical resection followed by chemotherapy and radiation therapy. Unfortunately, this multimodal approach still translates to a mean survival of only 12 to 14 months. Gliomas cannot be cured.

One desirable approach to managing this devastating cancer would be to inhibit malignant glial cell (MGC) invasion. Maintaining MGCs in a local environment leaves further treatment options open. However, there are currently no therapeutic strategies available for the inhibition of brain cancer invasion.

Many molecules have been implicated in malignant glioma invasion, however the molecular mechanisms underlying the process are not well understood. The current understanding of cell invasion is a composite derived from studies of different cell types and environments. Cell invasion involves the extension of a cellular process, attachment through focal adhesion (FA) formation, degradation of the extracellular matrix to create space to accommodate the moving cell, translocation of the cell body forward, and release of cell rear FAs (Friedl and Wolf, Nat Rev Cancer 3:362-74, 2003). This multistep process requires the coordinated action of cell surface receptors, signaling pathways, cytoskeletal elements, FA components, and extracellular matrix degrading enzymes (Burridge and Chrzanowska- Wodnicka, Annu. Rev. Cell. Dev. Biol. 12:463-519, 1996; Lauffenburger and Horwitz, Cell 84:359-369, 1996; Ridley et al. , Science 302: 1704-9, 2003). Within this scheme, recent studies have pointed to the importance of actin/microtubule (MT) dynamics in both cell front membrane protrusion and cell rear retraction (Palazzo and Gundersen, Sci. STKE 2002:PE31 , 2002; Rodriguez et al. , Nature Cell Biology 7:599-609, 2003). Cell rear retraction requires regulated FA disassembly and the actin/MT system plays a key role in the process.

While there are many similarities between cell movement in normal physiologic conditions and in cancer, MGCs are thought to utilize additional or alternate mechanisms (Beadle et al., Mol Biol Cell. 19:3357-68,2008). Recent studies have suggested that MGCs invade the dense substance of the brain using a mode of cell movement that is similar to neural progenitor cell movement.

The epidermal growth factor receptor (EGFR)/phosphatidylinositol 3- kinase (PI3K)/Akt pathway is a major driver of GBM invasion (Fan and Weiss, Curr. Top. Microbiol. Immunol. 347:279-296, 2010). EGFR activates the lipid kinase PI3K which then phosphorylates PI(4,5)P₂ to form

PI(3,4,5)P₃. Phosphatase and tensin homolog (PTEN) reverses this process by dephosphorylating PI P₃ to PIP₂. PIP₃ binds the pleckstrin homology domain of Akt thereby recruiting it to the cell membrane. Once there, it is activated by phosphorylation at Thr308 and Ser473 by PDK1 and mTORC2, respectively (Hers et al., Cell. Signal. 23: 1515-1527, 201 1 ). Activated Akt translocates from the membrane to the cytosol and nucleus where it drives downstream pathways affecting cell proliferation, survival, metabolism and invasion (Manning and Cantley, Cell 129: 1261 -1274, 2007; Fan and Weiss, Curr. Top. Microbiol. Immunol. 347:279-296, 2010; Hers et al., Cell. Signal. 23: 1515-1527, 201 1 ).

Oncogenic alterations of EGFR, PIK3CA, and PTEN have been identified in GBM. Combined, this pathway is deregulated in over 80% of GBMs and elevated phosphorylated Akt levels are found in the majority of GBMs. Thus, EGFR is an attractive therapeutic target, but clinical trials testing EGFR inhibitors for GBM have been disappointing (Brandes et al., Clin. Cancer Res. 14: 957-960, 2008; Van den Bent et al., J. Clin. Oncol. 27: 1268-1274, 2009; Brown et al., J. Clin. Oncol., 26: 5603-5609, 2008).

Akt can also be activated independent of receptor tyrosine kinase

(RTK) or PI3K activity. The viral oncogene v-akt is created by the addition of a myristoylation signal to the amino terminus of Akt. This allows Akt to associate with the cell membrane becoming constitutively active, bypassing the need for upstream RTK or PI3K involvement (Andejelkovic et al., J. Biol. Chem. 272: 31515-31524, 1997; Ahmed et al., Oncogene 7: 1957-1963, 1993). Similarly, mutation in the pleckstrin homology domain of Akt leads to association of Akt with the cell membrane and constitutive activation in breast, colorectal and ovarian cancers (Carpten et al., Nature 448: 439-444, 2007). Thus, events promoting Akt localization to the cell membrane can be sufficient for its activation.

The "down regulated in renal cell carcinoma (DRR1 )" gene (also known as TU3A, and referred to herein as DRR, DRR-1 and DRR1 interchangeably) was originally cloned from the short arm of chromosome 3 from patients with renal cell carcinoma (Wang et al., Genes Chromosomes & Cancer 27: 1 -10, 2000). Wang et al. reported that the gene showed significant loss of expression in renal cell carcinoma (RCC) cell lines, as well as in primary tumours, and that transfection of the gene into DRR negative cell lines resulted in growth suppression, suggesting a role as a tumour suppressor for DRR. The function of the DRR gene product is not known. The gene sequence predicts a protein of 144 amino acids with a nuclear localization signal and a coiled domain. A putative role for downregulation of DRR1 gene expression in glioma progression has also been suggested by van den Boom et al. (van den Boom et al., Int. J. Cancer 119: 2330-2338, 2006), who reported that DRR1 gene expression is reduced in glioblastomas as compared to diffuse astrocytomas. There is a need for inhibitors of brain cancer invasion and for new therapeutic approaches for the treatment of glioma and other invasive cancers. SUMMARY OF THE INVENTION

We report herein the identification of a protein, "down regulated in renal cell carcinoma" or "DRR", as a novel therapeutic target for the treatment of brain cancer and other metastatic or invasive cancers such as breast, prostate, squamous cell, lung, renal, or colon cancer. We show here that DRR is a novel regulator of brain cancer invasion. DRR drives MGC invasion in both in vitro and in vivo invasion assays, and DRR interaction with actin and microtubules (MTs) is essential for focal adhesion (FA)

disassembly and cell invasion. Moreover, DRR is not expressed in normal human brain glia, but is highly expressed in the invasive component of malignant gliomas, indicating a strong correlation between DRR expression in malignant gliomas and invasion. DRR also induces Akt phosphorylation and recruits Akt to focal adhesions. Further, antisense oligonucleotide- mediated ablation of DRR prevents tumor cell invasion in a mouse xenograft model. These findings are novel and unexpected, particularly in view of previous reports identifying a role for DRR as a tumour suppressor (Wang et al., Genes Chromosomes & Cancer 27: 1 -10, 2000; van den Boom et al., Int. J. Cancer 119: 2330-2338, 2006).

Taken together, our findings identify DRR as a novel regulator of cancer invasion, e.g., brain cancer invasion, and a target for therapeutic intervention in the treatment of metastatic or invasive cancers such as glioma, breast, prostate, squamous cell, lung, renal, or colon cancer.

Accordingly, there are provided herein compositions and methods for the treatment of cancer, e.g., glioma, comprising nucleic acid molecules effective at reducing the expression of DRR in tumor cells. The nucleic acid molecules of the invention include, for example, therapeutic RNAs such as antisense oligonucleotides, or short interfering RNAs (siRNA) molecules or vectors which encode antisense oligonucleotides or siRNAs. The siRNA molecules or the vectors that encode them are also referred to here as RNAi molecules. RNAi refers to "RNA interference", the process by which gene silencing is achieved by these siRNA molecules (Watts et al., Drug Discovery Today, 13: 842-855, 2008).

In an embodiment, there is provided herein a method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing a therapeutic RNAi or antisense molecule comprising the sequence of SEQ I D NOs: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21 /22 or 23/24, or a fragment or derivative thereof, to tumor cells, wherein the RNAi molecule or antisense molecule (e.g., antisense oligonucleotide or antisense RNA) reduces the expression of DRR in the tumor cells.

In another embodiment, there is provided a RNAi or antisense molecule for reducing the expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof.

In yet another embodiment, there is provided a method for reducing the expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing to tumor cells a DNA molecule comprising a sequence which encodes the sequence of SEQ I D NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21 /22 or 23/24, or a fragment or derivative thereof, wherein the DNA encodes a siRNA molecule or antisense molecule suitable for reducing the expression of DRR in the tumor cells. In some embodiments, the DNA molecule is inserted in an expression vector suitable for the production of dsRNA or suitable for the production of antisense RNA. The expression vector may, for example, comprise a sequence encoding the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof.

There are also provided herein methods of treating cancer comprising administering the RNAi or antisense molecules described herein or a vector that encodes siRNAs to a subject in need thereof. Methods of delaying the progression of cancer comprising administering the RNAi or antisense molecules described herein or a vector that encodes them to a subject in need thereof are also provided.

In some embodiments, the antisense and RNAi molecules and/or the vectors described herein may be used in combination with one or more cancer therapies selected from the group consisting of surgical resection, chemotherapy, radiation therapy, immunotherapy, and gene therapy.

In certain embodiments, the tumor cells are glioma cells, such as malignant glioma cells or glioblastoma cells. In some embodiments, the tumor cells are breast carcinoma cells, prostate carcinoma cells, squamous cell carcinoma cells, lung carcinoma cells, renal carcinoma cells, or colon carcinoma cells.

In an embodiment, there is a provided a pharmaceutical composition for the treatment of cancer comprising an RNAi or antisense molecule of the invention, or a vector that encodes the antisense molecule or RNAi molecule of the invention, and a pharmaceutically acceptable carrier. In one embodiment, the cancer is glioma, in particular malignant glioblastoma. In an embodiment, the cancer is metastatic or invasive breast, prostate, squamous cell, lung, renal, or colon cancer.

It will be appreciated by the skilled artisan that a vector will not encode a chemically modified siRNA directly, but rather produces short hairpin RNAs (shRNAs) which are subsequently processed by DICER to produce native siRNA duplexes that have sequences targeting the RNA of interest, e.g. , DRR mRNA. As used herein, reference to a vector or DNA "encoding a siRNA" refers to a vector or DNA producing a precursor RNA, e.g., a short hairpin RNA, which is processed to produce the siRNA.

In an embodiment, an "RNAi molecule" of the invention is an unmodified siRNA, e.g., produced by a vector in a cell in its native form. In another embodiment, an RNAi molecule of the invention is a chemically modified siRNA, e.g., a FANA-based molecule as described herein.

In another embodiment, there is provided a kit comprising the pharmaceutical compositions of the invention, and instructions for use thereof. The kits provided herein may further comprise a second active compound suitable for treating cancer, e.g., glioma, and/or for delaying the progression thereof, for simultaneous, separate or sequential administration to a subject.

The present invention also provides a method for enhancing efficacy of a cancer therapy for treatment of cancer, e.g., glioma, comprising administering an RNAi or antisense molecule of the invention or a vector that encodes the RNAi molecule or antisense molecule to a subject in need thereof, and simultaneously, separately or sequentially administrating a second cancer therapy. The second cancer therapy may be, for example, surgical resection, chemotherapy, radiation therapy, immunotherapy, and/or gene therapy.

Further provided herein is a method for inhibiting cancer cell invasion, e.g., malignant glial cell invasion, in a subject in need thereof, comprising providing to tumor cells an RNAi or antisense molecule of the invention, or a fragment or derivative thereof, wherein the RNAi or antisense molecule reduces the expression of DRR in the tumor cells. In an embodiment, malignant glial cell invasion is inhibited in a subject by providing to tumor cells a DNA molecule comprising the sequence encoding SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, wherein the DNA encodes an RNAi molecule or an antisense RNA suitable for reducing the expression of DRR in the tumor cells. In another embodiment, breast, prostate, squamous cell, lung, renal, or colon cancer cell invasion is inhibited in a subject by providing to tumor cells a DNA molecule comprising the sequence encoding SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, wherein the DNA encodes an RNAi molecule or an antisense RNA suitable for reducing the expression of DRR in the tumor cells.

In an embodiment, a method for diagnosis or prognosis of glioma in a subject, comprising measuring DRR expression in the glioma cells of the subject, wherein DRR expression indicates invasiveness of the cells, is provided. In another embodiment, a method for visualizing invasive glioma cells in a subject, comprising contacting glioma cells with a molecule which specifically binds DRR protein or mRNA and measuring DRR protein or mRNA levels in the cells, wherein cells which express DRR are invasive, is provided. In yet another embodiment, a method for diagnosis or prognosis of invasive cancer in a subject, comprising measuring DRR expression in the cancer cells of the subject, wherein DRR expression indicates invasiveness of the cells, is provided. In another embodiment, a method for visualizing invasive cancer or tumor cells in a subject, comprising contacting cancer or tumor cells with a molecule which specifically binds DRR protein or mRNA and measuring DRR protein or mRNA levels in the cells, wherein cells which express DRR are invasive, is provided. The invasive cancer may be, for example, breast, prostate, squamous cell, lung, renal, or colon cancer.

In yet another embodiment, there is provided a kit for diagnosis or prognosis of invasive cancer, e.g., invasive glioma, in a subject, comprising a detectably-labelled probe specific for DRR RNA or protein, a reporter means for detecting binding of the probe to the DRR RNA or protein, and instructions for use thereof.

In an embodiment, there is a provided a method for treating cancer comprising administering a therapeutic nucleic acid, e.g. an RNAi molecule or antisense molecule (e.g., a siRNA, an antisense oligonucleotide, or an antisense RNA), which reduces expression of DRR, or a vector encoding the therapeutic RNA to a subject in need thereof. In some embodiments, progression of cancer is delayed, malignant cell invasion is inhibited, and/or malignant glial cell invasion is inhibited. In an embodiment, the cancer is glioma, preferably malignant glioma, and more preferably glioblastoma. In another embodiment, the cancer is metastatic or invasive breast, prostate, squamous cell, lung, renal, or colon cancer. In an embodiment, the therapeutic RNA which reduces expression of DRR is complementary to or specifically hybridizes to DRR mRNA, or a fragment or derivative thereof.

It should be understood that the invention is not meant to be limited to the specific therapeutic nucleic acids recited herein; rather, use of any nucleic acid which functions to reduce expression of DRR in the compositions and methods of the invention is contemplated. In particular, therapeutic nucleic acids (e.g., RNAi molecules, siRNAs, antisense oligonucleotides, ribozymes, etc.) complementary to, or specifically hybridizing to a region of DRR, e.g. a region, fragment, portion or derivative of DRR mRNA, and which reduce expression of DRR, are encompassed. DNAs or vectors encoding the therapeutic RNAs of the invention are also encompassed herein. Nucleic acids aptamers, which are sequences that adopt a unique three-dimensional structure that recognizes (binds to) DRR through protein-nucleic acid interactions, are also encompassed herein.

In an embodiment, there is provided herein a siRNA molecule, wherein said siRNA molecule consists of: (a) a duplex region; and (b) either no overhang regions or at least one overhang region, wherein each overhang region contains six or fewer nucleotides, wherein the duplex region consists of a sense region and an antisense region, wherein said sense region and said antisense region together form said duplex region and each of said sense region and said antisense region is 18-30 nucleotides in length and said antisense region comprises a sequence that is the complement of SEQ ID NO: 4 or a fragment or portion thereof. In one embodiment, the antisense region and the sense region are each 19-25 nucleotides in length. In another embodiment, the antisense region and the sense region are each 21 nucleotides in length. The siRNA molecule may have at least one overhang region or may have no overhang regions. In an embodiment, the siRNA comprises one or more FANA nucleotides and/or one or more FRNA residues. In another embodiment, the siRNA comprises the sequence of siRNAI (SEQ ID NO: 17/18), siRNA2 (SEQ ID NO: 19/20) or siRNA3 (SEQ ID NO: 23/24). In yet another embodiment, the siRNA consists of the sequence of siRNAI , siRNA2 or siRNA3. In a further embodiment, the antisense region comprises a sequence that is complementary to nucleotides from position 227 to 245 of SEQ ID NO: 4. In a still further embodiment, DRR expression is downregulated by the siRNA.

There is also provided herein a siRNA molecule comprising a sense region and an antisense region that downregulates expression of a DRR gene via RNA interference (RNAi), wherein the sense region comprises a nucleotide sequence set forth in SEQ ID NO: 17, 19, or 23, and wherein the antisense region comprises a sequence that is complementary to a nucleotide sequence consisting of SEQ I D NO: 4 or a fragment or portion thereof. In an embodiment, the antisense region comprises a nucleotide sequence set forth in SEQ ID NO: 18, 20 or 24. In another embodiment, the antisense region consists of a nucleotide sequence which is set forth in SEQ ID NO: 18, 20 or 24. In yet another embodiment, the antisense region is complementary to nucleotides at positions 227-245 of SEQ ID NO: 4.

In another aspect, there is provided a recombinant nucleic acid construct or vector comprising a nucleic acid that is capable of directing transcription of a small interfering RNA (siRNA), the nucleic acid comprising: (a) at least one promoter; (b) a DNA polynucleotide segment that is operably linked to the promoter; (c) a linker sequence comprising at least 4 nucleotides operably linked to the DNA polynucleotide segment of (b); and (d) operably linked to the linker sequence a second polynucleotide, wherein the polynucleotide segment of (b) comprises a polynucleotide that is selected from the group consisting of SEQ ID NOs: 17, 19 and 23, wherein the second polynucleotide of (d) comprises a polynucleotide that is complementary to at least one polynucleotide that is selected from the group consisting of SEQ ID Nos: 17, 19 and 23. The DNA polynucleotide sequence may comprise SEQ ID NO: 17, 19 or 23 and/or the second polynucleotide may comprise SEQ ID NO: 18, 20 or 24. An isolated host cell transformed or transfected with a recombinant nucleic acid construct described herein is also provided.

In yet another aspect, there is provided an siRNA expression vector for downregulating expression of DRR in a subject in need therof, wherein the vector comprises: (1 ) a bacterial cassette comprising a bacterial origin of replication and a bacterial selectable marker M 1 ; (2) a cassette for selection in eukaryotic cells comprising a selectable marker M2 for eukaryotic cells, and in particular for mammalian cells, under the control of an appropriate promoter; (3) an siRNA transcription cassette comprising at least one region encoding an siRNA corresponding to a DRR gene, under control of regulatory elements for transcription in mammalian cells, which regulatory elements include at least one promoter capable of transcribing an siRNA in mammalian cells and a transcription terminator; wherein said siRNA transcription cassette is immediately downstream of the transcription initiation site or else a maximum of at most 20 base pairs away from the latter; said transcription initiation site being CCG and said siRNA transcription cassette comprising, downstream of the sequence encoding the siRNA, a transcription terminator which comprises a sequence of 6 consecutive thymidines, in the sense strand of the construct. In an embodiment, the DRR gene comprises the sequence set forth in SEQ ID NO:4 or a fragment or portion thereof, e.g., the fragment at positions 227-245 of SEQ ID NO: 4.

Also provided herein are pharmaceutical compositions comprising siRNAs or recombinant DNA constructs and vectors described herein, and a pharmaceutically acceptable carrier.

In a further aspect, there is provided herein an siRNA molecule, wherein said siRNA molecule consists of a duplex region, said duplex region consisting of a sense region and an antisense region, wherein: (a) said sense region and said antisense region together form said duplex region; (b) each of said sense region and said antisense region is 18-30 nucleotides in length; and (c) said antisense region comprises a sequence that is complementary to a nucleotide sequence consisting of SEQ ID NO: 4 or a fragment or portion thereof. In an embodiment, the sequence of said antisense region is complementary to a sequence comprising nucleotides from position 227 to 245 of SEQ ID NO: 4. In another embodiment, the sequence of said antisense region is complementary to a sequence consisting of nucleotides from position 227 to 245 of SEQ ID NO: 4. The antisense region and sense region may each be, e.g., 19-25 nucleotides in length.

In an embodiment, the sense region comprises the sequence set forth in SEQ ID NO: 17, 19 or 23 and the antisense region comprises the sequence set forth in SEQ ID NO: 18, 20 or 24. In another embodiment, the siRNA molecule consists of a duplex comprising the sequence set forth in SEQ ID NO: 17/18, 19/20 or 23/24. In yet another embodiment, the siRNA molecule consists of a duplex consisting of the sequence set forth in SEQ ID NO: 17/18, 19/20 or 23/24.

In other embodiments, the siRNA molecules provided herein comprise one or more FANA nucleotides and/or one or more FRNA residues.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, an embodiment or embodiments thereof, and in which:

Fig. 1 shows the validation of DRR as a regulator of invasion, wherein:

(A) shows and outline of a functional genetic screening assay; (B) shows a mixed tumor spheroid containing WT glial cells (cytotracker red label) and DRR overexpressing cells (DRR⁺, transparent) showing hyperinvasion of DRR⁺ cells; solid circle demarcates invasion front of WT cells, and dashed circle demarcates invasion front of DRR⁺ cells; (C) shows control mixed tumor spheroid showing equal invasion of WT cytotracker red labeled cells and WT unlabelled cells demonstrating that cytotracker red labeling does not influence invasion; (D) shows quantitative analysis of invasion; (E) shows quantification of maximal invasion of WT- (red bars) and DRR⁺- (empty bars) cells; data are mean ± s.e.m. (n = 14 for each cell line); asterisk, P < 0.001 ; (F) shows tumor spheroid generated from DRR^" cells, wherein circle demarcates invasion front; (G) shows tumor spheroid generated from WT cells, wherein circle demarcates invasion front; (H) shows high magnification image of inset in (F), showing that DRR^" cells have a round cell shape; (I) shows high magnification image of inset in (G), showing that WT cells have an elongated cell shape; (J) shows quantification of cell invasion comparing DRR^" cells and WT cells; Cells invading greater than 400 μιη were counted; data are mean ± s.e.m. (n = 8 for each cell line); asterisk, P < 0.001 ; (K) shows quantification of the effect of DRR expression on cell shape showing that DRR expression promotes an elongated cell shape; (L) shows DRR⁺ cells implanted into mouse brain showing elongated cell shape and invasion into corpus callosum (cc), wherein arrows indicate MGCs that have invaded the corpus callosum, arrowheads delineate tumor border, arrow in inset indicates tumor implantation site, bar = 100 μιη; (M) shows DRR^" cells implanted into mouse brain showing round cell shape and no evidence of invasion towards the corpus callosum, wherein arrowheads delineate tumor border and arrow in inset indicates tumor implantation site, bar = 100 μιη; and (N) shows quantification of cell proliferation in DRR⁺, WT, and DRR^" cells.

Fig. 2 shows that DRR is expressed in neurons and human gliomas but not in normal glia. DRR immunolabeling of normal human brain at low (A and B) and high (C and D) magnification shows that DRR is found within the cortex but not in white matter (wm). Expression of the glial marker GFAP does not overlap with DRR (A-D). DRR is not expressed in the aneuronal molecular layer (ml) of the cortex (C). High magnification imaging shows that DRR is highly expressed in neurons (E) but not in white matter (F). Rat brain cultures similarly show that DRR expression overlaps with the neuronal marker MAP2 in neurons (G-l) but not with the glial marker GFAP in glia (J- L). In (M), DRR expression in eight malignant gliomas of each grade was assessed. Both grade 2 and grade 3 gliomas (left panels, top and bottom) uniformly express high levels of DRR. In contrast, only the invasive peripheral tumor (PT) portions of grade 4 gliomas uniformly express DRR (right panel, bottom). The central tumor (CT) portion exhibits variable DRR expression, negative in 5 and positive in 3 tumors (right panel, top and middle). H & E: hematoxylin and eosin, Ki-67: marker of cell division revealing high levels of proliferation in the central tumor region.

Fig. 3 shows that DRR associates with the actin cytoskeleton and interacts with LC2. (A) shows that transfected DRR localizes along actin stress fibers and focal adhesions. Arrows indicate expression at FA sites. Actin is labeled with phalloidin. The non-actin binding DRR^APEPE, and the non-LC2 binding DRR^AHRE, are diffusely expressed in the cytoplasm. They do not localize to actin or FAs. DRR^AHRE can also be found in the nucleus. (B) shows co-localization of FLAG-DRR and MYC-LC2 along actin stress fibres, lamellipodia and membrane ruffles. (C) shows co-immunoprecipitation of heterologously expressed FLAG-DRR and MYC-LC2 from glial cells. MYC- LC2 co-immunoprecipitates with FLAG-DRR and FLAG-DRR^APEPE but not when the conserved N-terminal HRE sequence, DRR^AHRE, is mutated.

Fig. 4 shows that DRR association with actin and LC2 is required for cell invasion. (A) shows 3D invasion assays of WT, DRR⁺, DRR^APEPE and DRR^AHRE in a 3D collagen matrix. (B) shows a closer view of the spheroid margins showing cell invasion. Asterisk indicates the spheroid edge in DRR^APEPE cells. (C) Quantitative analysis of cell invasion after 24, 48 and 72h.

Fig. 5 shows that DRR promotes focal adhesion dynamics. DRR⁺ and

WT cells were transfected with GFP-paxillin and imaged using confocal videomicroscopy for 170 minutes at 1 minute intervals. (A) shows DRR⁺ cells transfected with GFP-paxillin. Representative cell showing dynamic membrane protrusions and FA assembly and disassembly. Arrows indicate areas of robust FA assembly and disassembly. Boxes, b and c, represent high magnification areas shown in (B) and (C). (D) shows WT cell transfected with GFP-paxillin. Representative cell showing a lack of membrane protrusions and stable FAs. No FAs were identified that assembled or disassembled over the imaging interval.

Fig. 6 shows that DRR promotes focal adhesion disassembly. DRR^" ,

DRR⁺ (A) and DRR^APEPE (B) were starved for 24h and left untreated or treated for 4h with 10μΜ nocodazole. The MT depolymerizer was then washed out for the indicated time. DRR expression promotes FA

disassembly whereas DRR deficiency leads to more stable FAs.

Fig. 7 shows that DRR organizes the actin and microtubular cytoskeletons. DRR⁺ (A) and DRR^" (B) cells were grown on FN (^g/ml) for 48h before fixation. Cells were then labeled for MTs (green), actin (red) and vinculin (blue). The insets represent higher magnification of the indicated outlined boxes. Arrows indicate that MTs are targeted to FAs in DRR⁺ cells, whereas MTs do not reach FAs in DRR^" cells. Bars = 20 μΐη. (C) A working model summarizing the role of DRR in cytoskeletal organization and invasion. We propose that with LC2, DRR acts as an actin-MT crosslinker.

DRR targets MTs to FAs promoting their disassembly, cell rear retraction, and cell invasion.

Fig. 8 shows DRR protein expression in DRR^" and DRR⁺ stable cell lines. (A) shows protein blotting showing increased DRR expression in the

DRR⁺ cell line and reduced DRR expression in the DRR^" cell line in comparison to wild-type cells. (B) shows DRR⁺ cells implanted into mouse brain showing elongated cell shape and invasion into corpus callosum (cc).

Arrows indicate MGCs that have invaded the corpus callosum. (C) shows DRR^" cells implanted into mouse brain showing round cell shape and no evidence of invasion towards the corpus callosum. Arrowheads delineate tumor border. Bars = 100 μιη.

Fig. 9 shows that DRR regulates the morphology of migrating cells.

The morphology of migrating DRR⁺, WT and DRR^" cells was assessed in a 2D migration assay. Representative images captured over a 10 hour interval show that DRR⁺ cells extend long thin protrusions whereas WT and DRR^" cells migrate using broad lamella.

Fig. 10 shows DRR expression in human cortex. In (A) DRR immunolabelling of normal human brain cortex at high magnification shows that DRR is not expressed in the aneuronal molecular layer (ml). In (B) adjacent section GFAP immunolabelling shows the presence of astrocytes in the molecular layer (arrows) which are DRR negative.

Fig. 11 shows that DRR regulates focal adhesion dynamics and invasion in multiple glioma cell lines. Control U343 or U343-DRR^" cells (A) and control C6 or C6-DRR^" cells (B) were colabeled for actin (phalloidin) and

FAs (vinculin). Control cells contain small FAs whereas cells with reduced

DRR expression contain large FAs. Bars = 20 μΐη. Reduced DRR

expression in U343 (C), C6 (D), and U87 (E) glioma cells leads to a significant reduction in invasiveness in a 3D invasion assay.

Fig. 12 shows localization of endogenous DRR in malignant glial cells.

Immunolabeling wild-type U251 cells with the anti-DRR antibody reveals localization along actin stress fibers, FAs, membrane ruffles, and in the nucleus.

Fig. 13 shows a comparison of amino acid sequences within regions required for DRR-actin association across species.

Fig. 14 shows truncation analysis to identify DRR regions required for stress fibre localization. dsRed was fused to the C-terminus of full length and truncated versions of DRR. The DRR-dsRED fusion proteins were expressed in WT U251 and assayed for stress fibre localization. These data show that amino acids 62-100 and 108-120 are required for stress fibre localization.

Fig. 15 shows that DRR reduction using RNA interference leads to specific on-target effects on focal adhesion dynamics. (A) shows U251 cells expressing GFP-RNAi targeting DRR, (B) shows DRR rescue cell transiently expressing DRR as identified by immunolabeling DRR (arrow), and in (C) FAs were visualized by immunolabeling vinculin. DRR⁺ FA phenotype (reduced FA size and increased FA number) can be rescued by expressing DRR in DRR^" cells.

Fig. 16 shows that reduction of DRR expression inhibits human glioma invasion. Human high grade gliomas were surgically resected and immediately placed in culture. Two weeks later they were transfected with a control GFP vector or DRR-RNAi (vector also contains GFP). Tumor spheroids were generated from these cells and implanted into a collagen matrix. Brightfield (upper lanes) and fluorescence images (lower lanes) were captured at 1 to 14 days post-implantation. Non-transfected tumors (A) and control GFP-transfected tumors (B) readily invade, whereas DRR-RNAi transfected tumors (C) do not. (D) shows quantification of invasion distance from spheroid edge, wherein D indicates days, GFP is green fluorescent protein and GBM is glioblastoma (high grade glioma).

Fig. 17 shows comparison of efficacy of different DRR antisense oligonucleotides in reducing DRR expression. DRR+ cells were transfected with the indicated DRR antisense (Antisense 4 (SEQ ID NO: 14), Antisense 5 (SEQ ID NO: 15) or Antisense 6 (SEQ ID NO: 16); a non-targeting control antisense (Ctl Antisense); or left untransfected (Untransfected). 72 hours post-transfection, cells were lysed and analysed using 12% SDS-PAGE. DRR expression level was detected with anti-DRR antibody. Western blot of tubulin is included as loading control.

Fig. 18 shows visualization of changes in DRR actin's cytoskeletal and focal adhesion. Using lipofectamine 2000 reagent, DRR+ cells were transfected with a non-targeting control antisense (ctl Antisense), the indicated DRR antisense (Antisense G4 (SEQ ID NO: 14), Antisense G5 (SEQ ID NO: 15) or Antisense G6 (SEQ ID NO: 16)) or left untransfected (Untransfected). At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (left column; green) and actin (right column; red).

Fig. 19 shows analysis of DRR+ cell migration using an in vitro scratch assay. DRR+ cells were untransfected (CTL) or transfected with a non- targeting control antisense (Ctl Antisense) or transfected with the indicated antisense (antisense G4 (SEQ I D NO: 14), antisense G5 (SEQ ID NO: 15) or antisense G6 (SEQ ID NO: 16)). (A): Images of the scratch were acquired at 0, 24 hours and 48 hours; (B): Quantitative analysis of cell migration is shown; DRRCTL1 and DRRCTL2 represent untransfected, DRR+ controls. Green bar (top bar): 48 h; Red bar (center bar): 24 h; Blue bar (bottom bar): 0 h.

Fig. 20 shows analysis of DRR+ cell invasion using an in vitro 3D invasion assay. DRR+ cells were untransfected (CTL) or transfected with indicated antisense G6 (SEQ ID NO: 16). (A): Images of cell invasion were acquired at 0, 24, 48, 72 and 96 hours; G6 (sphere#1 )and G6b (sphere#2) are two separate examples of tumors treated with antisense G6. (B):

Quantitative analysis of invasion is shown, where the green bars (the lefthand bar of each pair of bars) indicate CTL (control) and the red bars (th righthand bar of each pair of bars) indicate antisense G6 (SEQ ID NO: 16).

Fig. 21 shows visualization of changes in GBM6 actin's cytoskeletal and focal adhesion. GBM6 cells were transfected with indicated antisense (Untransfected; Ctl antisense; or antisense G6 (SEQ ID NO: 16) using lipofectamine 2000 reagent. At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (green; left column) and actin (red; right column).

Fig. 22 shows analysis of GBM6 cell migration using an in vitro scratch assay. GBM6 cells were transfected with the indicated antisense (Antisense G4 (SEQ ID NO: 14), Antisense G5 (SEQ ID NO: 15), or

Antisense G6 (SEQ ID NO: 16)) or a non-targeting control antisense (CTL Antisense) acquired at 0 and 24 hours.

Fig. 23 shows comparison of efficacy of different DRR siRNA oligonucleotides in reducing DRR expression. DRR+ cells were transfected with the indicated DRR siRNA (siRNAI (SEQ ID NO: 17/18), siRNA2 (SEQ ID NO: 19/20) or siRNA3 (SEQ ID NO: 23/24); a non-targeting control siRNA (Ctl siRNA; SEQ ID NO: 25/26); or left untransfected (Untransfected). 72 hours post-transfection, cells were lysed and analysed using 12% SDS- PAGE. DRR expression level was detected using an anti-DRR antibody. Western blot of tubulin is included as a loading control.

Fig. 24 shows comparison of efficacy of different DRR siRNA oligonucleotides in reducing DRR expression. MNI 1 stem cells were transfected with the indicated DRR siRNA (siRNAI (SEQ ID NO: 17/18), siRNA2 (SEQ ID NO: 19/20) or siRNA3 (SEQ ID NO: 23/24); a non-targeting control siRNA (Ctl siRNA; SEQ ID NO: 25/26); or left untransfected

(Untransfected). 72 hours post-transfection, cells were lysed and analysed using 12% SDS-PAGE. DRR expression level was detected using an anti- DRR antibody. Western blot of tubulin is included as a loading control.

Fig. 25 shows changes in actin cytoskeletal and focal adhesions when DRR expression is reduced. In (A), DRR+ cells were transfected with a non- targeting control siRNA (ctl siRNA), the indicated DRR siRNA (siRNAI (SEQ ID NO: 17/18), and siRNA2 (SEQ ID NO: 19/20) or left untransfected (Untransfected). siRNA2 was fluorescently labelled with cy5 (SEQ ID NO: 21 /22) and transfected into DRR+ cells. At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (left column; green) and actin (right column; red). (B) shows visualization of vinculin (left panel; green), actin (center panel; red) and siRNA (right panel; blue).

Fig. 26 shows changes in actin cytoskeletal and focal adhesions when DRR expression is reduced in GBM6 cells. GBM6 cells were transfected with a non-targeting control siRNA (ctl siRNA), the indicated DRR siRNAI (SEQ ID NO: 17/18), or left untransfected (Untransfected). At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (left column; green), and actin (right column; red).

Fig. 27 shows extent of human glioblastoma cell migration following reduction of DRR expression using an in vitro scratch assay for observing cell invasiveness. GBM6 glioma cells were "scratched" to clear cells from an area of the plate; the ability of the plated GBM6 glioma cells to "invade" back into the cleared area was monitored over time. GBM6 cells were transfected with the indicated siRNAs or left untransfected (control). Images of the scratch were acquired at 0, 24, 48, 72, and 96 hours.

Fig. 28 shows changes in actin cytoskeletal and focal adhesions when malignant glioma cells are treated with DRR targeting siRNA. The top row shows malignant glioma cells (Ctl); they are elongated, and lack strong focal adhesions at the surface. The middle row shows malignant glioma cells treated with siRNAI (SEQ ID NO: 17/18; referred to in the figure as D1 ).

Once treated with siRNAI , the cells morphologically resemble a more typical cell, rounded and with strong focal adhesions. The bottom row shows malignant glioma cells treated with Cy5-siRNA2 (SEQ ID NO: 21/22; referred to in the figure as D2-Cy5). The green dye (vinculin) stains focal adhesions; the red dye (phalloidin) stains actin (the cytoskeleton of the cell); for D2-Cy5, siRNA is labelled with a Cy5 dye (blue), which allows the location of the siRNAs to be visualized within the cells (blue). In each row, the left panel shows visualization of vinculin (green); the second panel from the left shows visualization of actin (red); the third panel from the left shows visualization of siRNA (blue); and the right-most panel shows visualization of all three stains.

Fig. 29 shows Phospho-Akt is elevated in DRR over-expressing cells, (a): DRRov cells co-labelled with vinculin (green) and DRR (red); (b): Western blot showing the expression of DRR, Serine 473 and Threonine 308 Akt phosphorylation, Akt, NFkB, pGsk3, Gsk3 in DRRov and CTL cells.

Tubulin is shown as a loading control; (c): Quantification of pAkt (Ser473) fluorescence intensity in DRRov and CTL cells. Total fluorescence intensity was normalized to control cells (n=3, S EM_DRR_OV=0.37); (d): Western blot showing decreased DRR expression level using AOs in DRRov cells correlates with decreased pAkt expression level. Tubulin is shown as a loading control.

Fig. 30 shows that high expression level of pAkt in DRRov is EGFR- independent, (a): Western blot showing phosphotyrosine (pTyr) levels in untreated (unt) and EGF (50ng/mL) treated lysates showed highest levels in DRRov compared to CTL. This agrees with the upregulated EGFR expression in DRRov cells; (b): EGFR Immunolabeling in DRRov and CTL; (c): Western blot showing pAkt expression levels in response to EGF

(50ng/mL) treatment and EGFR inhibition with AG1478 (1 μΜ) in DRRov. Phospho-Akt in DRRov was insensitive to EGFR inhibition. For all westerns, tubulin is shown as a loading control; (d): 3D invasion assay of DRRov cells untreated or treated with AG1478 (1 μΜ); (e): Quantification of invasion at 24, 48 and 72 hours (n=3, Two-way ANOVA, p>0.05). EGFR inhibition did not significantly reduce DRR-driven invasion.

Fig. 31 shows Western blots showing: (a): pAkt expression level in DRRov and CTL cells untreated or treated with U0126 inhibitor or with DMSO in the absence or presence of EGF (50ng/ml); (b): pAkt level in response to Rho inhibitor, C3 transferase ^g/mL) treatment in DRRov or CTL; (c): Phospho-Akt levels in DRRov transfected with siRNA (si) against integrin- linked kinase (ILK) [or scramble (scr)] at 100 and 120nM after 48 and 72 hours post-transfection; (d): Phospho-Akt and total Akt levels in response to the SFK inhibitor PP2 (5μΜ) and its inactive analog PP3 (5μΜ) treatment in DRRov and CTL cells; (e): pAkt and Akt levels in response to PP2 (10μΜ) treatment in DRRov and DRRkd after being plated on fibronectin (FN; 50 μ/ml) for 30 minutes or 4 hours. pFAK and Fak levels are shown to verify FN efficacy; (f): pFak and pAkt levels after FAK inhibition with PF228 of DRRov or CTL cells plated on fibronectin (FN; 50 g/ml); (g): Phospho-Akt levels in response to PI3K inhibition with LY294002 (at 5, 10 and 20μΜ) in the absence or presence of EGF (50ng/ml). Phosphotyrosine (pTyr) levels are shown to verify EGF stimulation. DMSO is a vehicle control; (h): Phospho-Akt levels in response to LY294002 (LY) (5μΜ) in DRRov after being plated on fibronectin (FN; 50 μg/ml) for 30 minutes or 1 .5 hours. Phospho-FAK (pFAK) levels are shown to verify FN treatment. For all westerns, tubulin is shown as a loading control.

Fig. 32 shows that Phospho-Akt signaling is cell-adhesion dependent and DRR recruits Akt to the focal adhesion, (a): Western blot showing in

DRRov and CTL lane 1 -cells plated on fibronectin (FN; 50 μg/ml), lane 2-cells in the non-adherent (non-adh) fraction after RGD treatment, lane 3-cells in the adherent (adh) fraction after RGD treatment, lane 4-cells in suspension without RGD treatment. Phospho-Akt levels decreased in non-adherent cells; (b): Quantification of pAkt band densitometry corrected for loading control and measured relative to the fibronectin (FN) condition in DRRov (n=3, Oneway ANOVA, Bonferroni p<0.05, SEM_RGDnon-adh=0.1 , SEM_RGDadh=0.1 ,

SEM_Suspension=0.002); (c): Immunolabeling of Akt (green) or pAkt (green) with the focal adhesion marker, vinculin (red) in DRRov and CTL cells that have been plated on fibronectin for 30 min or 6 hours; (d): Co-staining of pAkt (green) and vinculin (red) in DRRov cells treated with PP2 (10μΜ) or left untreated where pAkt localization at focal adhesion area is lost when treated with SFK inhibitor PP2; (e): Immunolabeling of pAkt (green) and vinculin (red) in DRR^APEPE showing that pAkt is no longer at the focal adhesion when DRR is mutated; (f): Western blot showing a reduction in pAkt expression levels in the DRR-mutant cell lines, DRR^APEPE, in comparison with DRRov cells.

Fig. 33 shows that SFK and PI3K inhibition prevent invasion of DRR- overexpressing cells, (a): 3D invasion assays of DRRov and CTL cells untreated or treated with PP2 or LY294002 at time 0 and 48 hours; (b):

Quantification of 3D invasion of DRRov after 48 hours with indicated treatment (n=3, Two-way ANOVA, Bonferroni **p<0.01 , ***p<0.001 ,

****p<0.0001 , SEM_unt=7.3, 1 1 ,2, 13.7,

25.2, SEM_PP2=3.9, 2.8, 2.9); (c): Quantification of invasion of CTL following 48 hours with indicated treatment (n=3, Two-way ANOVA, Bonferroni ***p<0.001 ,

****p<0.0001 96hrs, SEM_unt=32.8,

SEM_PP2=13.2); (d): Quantification of 3D invasion of DRRov untreated or treated with a combination of LY294002 and PP2(conc) over 96 hours (n=3, Two-way ANOVA, Bonferroni ****p<0.0001 , SEM_unt=2.5, 9.6, 15.1 , 22.2,

S

2.7, 4.0, 2.8); (e): Quantification of the relative fold difference in invasion between DRRov untreated (unt) and singly treated with either LY294002 (LY) or PP2, or treated with the combination of both

(combo) (n=3, One-way ANOVA, Bonferroni p<0.05, SEM_LY=5.5,

SEMpp₂=0.4, SEM_combo=2.1 ).

Fig. 34 shows DRR as a therapeutic target for brain cancer invasion, (a): Western blot showing DRR expression in human glioma stem cells, left: untransfected (0), transfected with control non-targeting antisense oligonucleotide (scr) or with DRR targeting antisense oligonucleotide (AO). Tubulin is shown as a loading control, (b): Mouse brain section stained with H&E, human Sox-2 to identify the injected cells (red), cy5 fluorescence showing AOs (green) and merged images of AOs with Sox-2 are shown (merge). Arrow heads indicate invading cells; src: control non-targeting antisense oligonucleotide; AO: DRR targeting antisense oligonucleotide.

Fig. 35 shows fold change in normalized DRR mRNA expression for the indicated tissue or tumour samples. Normalized expression of DRR was calculated by taking the relative quantity (DRR) divided by the relative quantity of a reference gene (HS14) and graphed as fold change expression. Increased DRR expression is correlated with invasiveness in breast, prostate and squamous cell carcinoma.

Fig. 36 shows a comparison of different DRR antisense efficacy in reducing DRR expression. DRR+ cells were transfected with 20 nM of indicated DRR antisense oligonucleotides (AONs) using Lipofectamine 2000, and DRR expression level was assessed following 72 hours post- transfection. There is shown a Western Blot of DRR+ cells transfected with the indicated DRR antisense (G5 (SEQ ID NO: 15), G6 (SEQ ID NO: 16)), a non-targeting control antisense (G1 (scramble) (SEQ I D NO: 1 1 ) or left untransfected (0). 72h post-transfection, cells were lysed and analysed in 12% SDS-Page. DRR expression level was detected with anti-DRR antibody. Western blot of tubulin is included as loading control.

Fig. 37 shows visualization of changes in DRR actin's cytoskeletal and focal adhesion. Using Lipofectamine 2000 reagent, DRR+ cells were transfected with a non-targeting control antisense (G1 ), the indicated DRR antisense (G5, G6) or left untransfected (not shown). At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (green) and actin (red). The third panel from the left shows the blue channel; nothing is visualized since the antisense oligonucleotides were not tagged with cy5 in this experiment. The right panel shows a merge of the vinculin and actin panels.

Fig. 38 shows tagged DRR antisense oligonucleotides efficiently reduce DRR levels. There is shown a western blot showing a DsredDRR stable cell line transfected with the indicated antisense oligonucleotide or untransfected (CTL). 72h post-transfection, cells were lysed and analysed in 12% SDS-Page. Anti-DRR (top) or anti-Dsred (bottom) antibodies were used to detect DRR expression levels. Tubulin was included as loading control. Quantification indicated significant decrease in DRR level in cells transfected with G5-Cy5, G6-Cy5, G5 or G6.

Fig. 39 shows reduction of DRR and larger focal adhesion observed with the expression of targeted DRR antisense. DsredDRR stable cells were transfected with the indicated Cy5-tagged antisense. 72h post-transfection cells were fixed and labelled with vinculin (green) to visualize focal adhesion. DRR expression level could be directly detected in the red channel

(dsredDRR) and antisense expression was detected in the blue channel. DsredDRR could no longer be detected in cells highly expressing G5-cy5 or G6-cy5 antisense, while non-targeting G1 -cy5 antisense did not affect dsredDRR levels. The far right panel shows a merge images of the 3 channels in the other 3 panels. Fig. 40 shows analysis of DRR+ cell migration by in vitro scratch assay. DRR+ cells were left untransfected (0), or transfected with a non- targeting control antisense oligonucleotide (G1 ) or the indicated antisense oligonucleotide (G5, G6). Cell migration was assessed at Oh, 24h and 48h. (A): Images of the scratch were acquired at Oh and 48h; (B): Quantitative analysis of cell migration is shown.

Fig. 41 shows that DRR silencing with G6-cy5 antisense showed reduced invasion in comparison with random antisense G1 -cy5. Analysis of DRR+ cell invasion was done by in vitro 3D invasion assay. (A): Western blot of DRR+ cells untransfected (0) or transfected with the indicated antisense (G6, G6-cy5, G1 -cy5) is shown; cell lysates were analyzed 72h post- transfection. Drr expression was detected with an anti-DRR antibody. Tubulin was included as loading control. (B): Cell invasion images of DRR+ cells expressing G1 -cy5 or G6-cy5 (blue cells) are shown. Cell invasions were acquired at day 1 , 2, and 6 with a 20x objective. Blue cells expressing G6- cy5 did not invade as far as G1 -cy5 expressing cells. (C): Quantitative analysis of invasion is shown at day 0 (dO), day 1 (d1 ), day 2 (d2), day 3 (d3), day 4 (d4), day 6 (d6) and day 8 (d8).

Fig. 42 shows antisense G5 and G6 successfully inhibit human glioma stem cell invasion. (A): 3D in vitro invasion images of human glioma stem cells left untransfected (CTL), transfected with control non-targeting antisense (G1 ) or with DRR targeting antisense (G5 or G6), as indicated, captured at different intervals (day 2, 6, or 9) with a 5x objective. (B):

Western blot showing DRR expression in human glioma stem cells left untransfected (CTL), transfected with control non-targeting antisense (G1 ) or with DRR targeting antisense (G5 or G6), as indicated, following 72h post- transfection. Tubulin is shown as a loading control.

Fig. 43 shows DRR targeted antisense prevent human glioma stem cell invasion in an in vivo mouse model. (A): There are shown mouse brain sections showing injected human glioma stem cells expressing control Cy5- non-targeting antisense (G1 -cy5) or DRR targeting antisense tagged with Cy5 (G5-cy5 or G6-cy5), as indicated. Human glioma stem cells expressing the antisense were directly detected with Cy5 fluorescence (left panel) and an H&E stained section was included to show tumor mass (middle panel; HNE). Merge images of G1 -cy5 expressing cells with H&E images (right panel) show invasion of human glioma stem cells far from the tumor mass, whereas G5 or G6 expressing cells are restricted within the tumor mass (right panel for second and third rows, respectively). (B) shows the same thing as (A), with cy5-tagged antisense shown in blue. We also observed a stronger signal in cells expressing G5-cy5, an indication of long-lasting effects of the antisense oligonucleotide when it is modified (FANA).

Fig. 44 shows a comparison of different DRR siRNA efficacy in reducing DRR expression. There is shown a Western blot analysis of DRR+ cells transfected with the indicated DRR siRNA, a non-targeting control siRNA (DRR4 siRNA (scramble)), or left untransfected (0). 72h post- transfection, cells were lysed and analysed in 12% SDS-Page. DRR expression level was detected with anti-DRR antibody. Western blot of tubulin is included as loading control. Results indicate that DRR1 siRNA, which is the unmodified siRNA, was the most efficient in decreasing DRR expression.

Fig. 45 shows visualization of changes in DRR actin's cytoskeletal and focal adhesion. DRR+ cells were transfected with the indicated siRNA (20 nM) using Lipofectamine 2000 reagent, or left untransfected. At 72 hours post-transfection, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (VINC; green) and actin (ACTIN; red). The last panel shows merged images. Reduction in DRR expression induced changes in cell morphology associated with decreased actin stress fibres, increased cortical actin and increases in focal adhesion size.

Fig. 46 shows visualization of changes in DRR actin's cyroskeletal and focal adhesion. DRR+ cells were transfected with siRNA2 fluorescently labelled with cy5. At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (green), actin (red), and siRNA (blue). Cells expressing DRR2 siRNA-cy5 showed large focal adhesion and increased in cortical actin. The right panel shows a merge images of the three other panels.

DETAILED DESCRIPTION

The present invention relates to the identification of downregulated in renal carcinoma (referred to herein as DRR, DRR1 or DRR-1 ) as a novel regulator of cancer, e.g., brain cancer, invasion and a target for therapeutic intervention in the treatment of invasive cancers, e.g., glioma, particularly malignant glioblastoma. In particular, there are provided herein novel compounds, pharmaceutical compositions and methods for inhibiting glioma tumor cell invasion and/or treating glioma comprising molecules which reduce the expression of DRR in glioma tumor cells. There are also provided herein novel compounds, pharmaceutical compositions and methods for treating metastatic or invasive cancers of any type, such as breast, prostate, skin (e.g., squamous cell carcinoma), lung, renal, or colon cancer.

The present invention thus provides compounds, in particular oligonucleotides and similar species, for use in modulating the function or effect of nucleic acid molecules encoding DRR. In some embodiments, this is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding DRR. A compound of this invention which hybridizes with its target nucleic acid is generally referred to as "antisense" and consequently, the mechanism of inhibition of DRR is referred to as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that the target RNA molecule is cleaved, degraded, or otherwise rendered inoperable. The present invention is concerned with targeting specific nucleic acid molecules which encode for DRR or a portion thereof, such as the mRNA encoding DRR.

As used herein, "hybridization" refers to the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid, e.g. DRR mRNA, interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise at least 80% sequence complementarity, at least 85% sequence complementarity, at least 90% sequence complementarity or at least 95% sequence complementarity to the target nucleic acid sequence. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol. 215: 403-410, 1990; Zhang and Madden, Genome Res. 7: 649-656, 1997).

In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. A non-limiting example of a hybridization condition is hybridization in 6x SSC buffer (900 mM sodium chloride containing 90 mM sodium citrate at pH 7. 0) containing 50% formamide, 0.5% sodium dodecyl sulfate and blocking reagents, carried out at 42°C for 16 hours. Membranes are then washed twice with Ix SSC containing 0.1 % SDS at room temperature, then O. Ix SSC containing 0. 1 % SDS at room temperature, and finally O. IxSSC containing 0.5% SDS at 42°C.

Antisense drugs are typically small (e.g.12-21 nucleotides, or 15-30 nucleotides) pieces of DNA or RNA that are chemically modified to engineer good drug properties. Antisense drugs work after binding (hybridizing) to a target RNA and forming a duplex. The formation of this duplex, or two- stranded molecule, prevents the RNA from functioning normally and/or from producing a protein. Antisense oligonucleotides inhibit mRNA translation via a number of alternative mechanisms including destruction of the target mRNA through RNaseH recruitment, interference with RNA processing or translation, nuclear export, folding or ribosome scanning. There are at least a dozen known antisense mechanisms that may be exploited once an antisense drug binds to its target RNA. For example, therapeutic RNAs may target non-coding RNAs, such as microRNAs, which are involved in the regulation of protein production within the cell. MicroRNAs are small naturally occurring RNA molecules that are created inside cells and appear to have critical functions in controlling processes or pathways of gene expression. There are nearly 700 microRNAs that have been identified in the human genome, and these are believed to regulate the expression of approximately one-third of all human genes. Other antisense drugs may for example control splicing, to favour production of one protein versus another.

Many different types of antisense oligonucleotides are known and may be used in the compositions and methods of the invention. It is contemplated that any of the known antisense technologies may be used to target DRR and reduce DRR expression. For example, oligonucleosides having alternating segments of sugar-modified nucleosides (e.g., 2'-0-modified ribonucleosides or arabinonucleosides) and 2'-deoxynucleosides, and/or oligonucleotides having alternating segments of sugar-modified nucleotides and 2'-deoxynucleotides, are known as "gapmers" and "altimers" and may be used for the preparation of antisense oligonucleotides. Altimers are described in, for example, PCT publication no. WO/2003/064441 , the contents of which are hereby incorporated by reference. In one embodiment, the therapeutic RNA of the invention is an antisense comprising an olignonucleoside comprising alternating segments of sugar- modified nucleosides and 2'-deoxynucleosides, wherein the segments or units each independently comprise at least one sugar-modified nucleoside or 2'-deoxynucleoside, respectively. For example, the oligonucleoside comprises alternating first and second segments, wherein the first segment comprises at least one sugar-modified nucleoside, and wherein the second segment comprises at least one 2'-deoxynucleoside. In embodiments, the oligonucleoside comprises at least 2 of each of the first and second segments thereby comprising at least 4 alternating segments.

In an embodiment, an oligonucleoside comprises an internucleoside linkage comprising a phosphate, thereby being an oligonucleotide. In embodiments the sugar-modified nucleosides and/or 2'-deoxynucleosides comprise a phosphate, thereby being sugar-modified nucleotides and/or 2'- deoxynucleotides. Thus in an embodiment, the invention provides an oligonucleotide comprising alternating segments or units of arabinonucleotides and 2'-deoxynucleotides, wherein said segments or units each independently comprise at least one arabinonucleotide or 2'- deoxynucleotide, respectively. In an embodiment, an oligonucleotide comprises at least 2 arabinonucleotide segments and at least 2 2'- deoxynucleotide segments thereby having at least 4 of the alternating units.

In an embodiment, a sugar-modified oligonucleotide is capable of adopting a DNA-like conformation. In an embodiment, a sugar-modified nucleotide is selected from the group consisting of arabinonucleotides, alpha- L-locked nucleic acids, cyclohexene nucleic acids, and ribonucleotides lacking an electronegative 2'-oxygen atom. In an embodiment, ribonucleotides lacking an electronegative 2'-oxygen atom are selected from the group consisting of 2'-alkyl-D-ribose and 2'-SCH₃-D-ribose.

In an embodiment, segments each independently comprise about 1 to about 6 arabinonucleotides or 2'- deoxynucleotides. In further embodiments, segments each independently comprise about 2 to about 5 or about 3 to about 4 arabinonucleotides or 2'-deoxynucleotides. In a further embodiment, segments each independently comprise about 3 arabinonucleotides or 2'- deoxynucleotides.

In an embodiment, an oligonucleotide has a structure selected from the group consisting of:

a) (A_x-D_y)_n I

c) (A_x-D_y)_m - A_x-D_y-A_x III

d) (D_y-A_x)_m- D_y-A_x- D_y IV

wherein each of m, x and y are each independently an integer greater than or equal to 1 , n is an integer greater than or equal to 2, A is an sugar-modified nucleotide and D is a 2'-deoxyribonucleotide.

In an embodiment, a sugar-modified nucleotide comprises a 2'substituent selected from the group consisting of fluorine, hydroxyl, amino, cyano, azido, -CH=CH₂,-C≡CH, alkyl, functionalized alkyl, alkoxy and functionalized alkoxy groups. In an embodiment, an alkyl group is a lower alkyl group. In an embodiment, a lower alkyl group is selected from the group consisting of methyl, ethyl and propyl groups. In an embodiment, a functionalized alkyl group is selected from the group consisting of methylamino, ethylamino and propylamino groups. In an embodiment, an alkoxy group is selected from the group consisting of methoxy, ethoxy and propoxy groups. In an embodiment, a functionalized alkoxy group is - 0(CH₂)_q-R, wherein q=2,3 or 4 and -R is selected from the group consisting of -NH₂,-OCH₃, and -OCH₂CH₃ groups.

In an embodiment, a sugar-modified nucleotide is an arabinonucleotide. In a further embodiment, a 2' substituent is fluorine and an arabinonucleotide is a 2'- fluoroarabinonucleotide (2'F-ANA ; also abbreviated "FANA").

In an embodiment, an antisense oligonucleotide of the invention comprises one or more internucleotide linkages selected from the group consisting of: a) phosphodiester; b) phosphotriester; c) phosphorothioate; d) phosphorodithioate; e) Rp-phosphorothioate ; f) Sp-phosphorothioate ; g) boranophosphate; h) methylene (methylimino) (3'CH₂-N (CH₃)-05'); i) 3'- thioformacetal (3'S-CH₂-05') j) amide (3'CH₂-C (O) NH-5'); k) methylphosphonate; I) phosphoramidate (3'-OP (0₂)-N5'); and m) any combination of (a) to (I).

In an embodiment, an antisense oligonucleotide consists of about 30 or fewer nucleotides, in a further embodiment, about 8 to about 25 nucleotides, and in yet a further embodiment, about 18 nucleotides. In an embodiment, an antisense oligonucleotide has about 12 nucleotides, about 15 nucleotides, about 18 nucleotides, about 20 nucleotides, about 25 nucleotides, or about 30 nucleotides. In another embodiment, an antisense oligonucleotide is from about 12 to about 30 nucleotides long.

In an embodiment, an antisense oligonucleotide has structure I wherein x=1 , y=1 and n=9, thereby having a structure: A-D-A-D-A-D-A-D-A- D-A-D-A-D-A-D-A-D. In an embodiment, an antisense oligonucleotide has structure II wherein x=1 , y=1 and n=9, thereby having a structure: D-A-D-A- D-A-D-A-D- A- D-A-D- A- D- A- D- A. In an embodiment, the above-mentioned oligonucleotide has structure III wherein x=2, y=2 and m=3, thereby having a structure: A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A. In an embodiment, the above-mentioned oligonucleotide has structure IV wherein x=2, y=2 and m=3, thereby having a structure: D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D. In an embodiment, the above-mentioned oligonucleotide has structure I wherein x=3, y=3 and n=3, thereby having a structure: A-A-A-D-D-D-A-A-A-D-D-D-A- A-A-D-D-D. In an embodiment, the above-mentioned oligonucleotide has structure II wherein x=3, y=3 and n=3, thereby having a structure: D-D-D-A- A-A-D-D-D-A-A-A-D-D-D-A-A-A. In an embodiment, the above-mentioned oligonucleotide has structure III wherein x=4, y=3 and m=1 , thereby having a structure: A-A-A-A-D-D-D-A-A-A-A-D-D-D-A-A-A-A. In an embodiment, the above-mentioned oligonucleotide has said structure IV wherein x=4, y=3 and m=1 , thereby having a structure: D-D- D-D- A- A- A- D-D- D-D- A- A- A- D-D- D-D.

In an embodiment, an antisense oligonucleoside further comprises a third segment comprising a modified nucleoside, wherein said third segment is adjacent to (a) the 5'end of said alternating first and second segments, (b) the 3'end of said alternating first and second segments, or (c) both (a) and (b).

In an embodiment, an antisense oligonucleotide further comprises a third segment comprising a modified nucleotide, wherein said third segment is adjacent to (a) the 5' end of said alternating first and second segments, (b) the 3' end of said alternating first and second segments, or (c) both (a) and (b). In an embodiment, a modified nucleotide is a modified ribonucleotide. In an embodiment, a modified ribonucleotide comprises a modification at its 2' position. In an embodiment, a 2' modification is selected from the group consisting of methoxy, methoxyethyl, fluoro and propylamino groups.

In an embodiment, an antisense oligonucleotide is an altimer comprising alternating segments of arabinonucleotide (ANA) such as 2'F- ANA (or FANA) and DNA. "Arabinonucleotide" as used herein refers to a nucleotide comprising an arabinofuranose sugar.

Preferably, in antisense molecules, there is a sufficient degree of complementarity to the target RNA (e.g., DRR) to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. A target RNA for antisense binding may include not only the information to encode a protein, but also associated ribonucleotides, which for example form the 5'- untranslated region, the 3'-untranslated region, the 5' cap region and intron/exon junction ribonucleotides.

Antisense molecules (oligonucleosides or oligonucleotides) of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, 3'-thioformacetal, amide, short chain alkyl or cycloalkyi intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with CH₂- NH-0-CH₂, CH₂-N (CH₃)-0-CH₂ (known as methylene (methylimino) or MMI backbone), CH₂-0-N (CH₃) -CH₂, CH₂-N (CH₃) -N (CH₃)-CH₂ and O- - N (CH₃) -CH₂-CH₂ backbones (where phosphodiester is O-P (0)₂-0- CH₂). In alternative embodiments, antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as "protein" or "peptide" nucleic acid) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (see for example, Nielsen et al., Science, 1991 ,254: 1497 and U. S. Pat. No. 5,539, 082). Phosphodiester bonds may be substituted with structures that are chiral and enantiomerically specific.

As noted above, oligonucleotides may also include species which include at least one modified nucleotide base.

Thus, purines and pyrimidines other than those normally found in nature may be used. As noted above, a nucleotide of a sugar-modified nucleotide segment (e. g. ANA segment) may comprise modifications on its pentofuranosyl portion.

Examples of such modifications are 2'-0-alkyl-and 2'- halogen- substituted nucleotides. Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH₃, F, OCN, O (CH₂)_n, NH₂ or O (CH₂) _n CH₃ where n is from 1 to about 10; Ci to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF₃; OCF₃; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 'SOCH₃ S0₂ CH₃; ON0₂; N0₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups of the nucleotide of the sugar-modified nucleotide segment may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.

"Nucleoside" refers to a base (e. g. a purine [e. g. A and G] or pyrimidine [e. g. C, 5-methyl-C, T and U] ) combined with a sugar (e. g. [deoxy] ribose, arabinose and derivatives). "Nucleotide" refers to a nucleoside having a phosphate group attached to its sugar moiety. In embodiments these structures may include various modifications, e. g. either in the base, sugar and/or phosphate moieties. "Modified nucleotide/nucleoside" as used herein refers to a nucleotide/nucleoside that differs from and thus excludes the defined native form.

"Oligonucleotide" as used herein refers to a sequence comprising a plurality of nucleotides joined together. An oligonucleotide may comprise modified structures in its backbone structure and/or in one or more of its component nucleotides. In embodiments, oligonucleotides of the invention are about 1 to 200 bases in length, in further embodiments from about 5 to about 50 bases, from about 8 to about 40 bases, and yet further embodiments, from about 12 to about 25 bases in length.

In an embodiment, a therapeutic RNA of the invention comprises an antisense RNA comprising a "gapmer". "Gapmers", which are also known as "chimeric antisense" oligos, are described for example in PCT international publication no. WO/2002/20773, the contents of which are hereby incorporated by reference.

For example, an antisense oligonucleotide may be a chimera constructed from arabinonucleotide or modified arabinonucleotide residues, flanking a series of deoxyribose nucleotide residues of variable length, that form a duplex with its target RNA sequence. Such resulting antisense oligonucleotide/RNA duplex is a substrate for RNaseH, an enzyme that recognizes this duplex and degrades the RNA target portion. RNaseH mediated cleavage of RNA targets is considered to be a major mechanism of action of antisense oligonucleotides.

In an embodiment, a therapeutic RNA is an antisense hybrid chimera, constructed from 2'-deoxy-2'-fluoro-B-D- arabinonucleotides (FANA) flanking a defined sequence constructed from R>-D-2'- deoxyribonucleotides (DNA). In one embodiment an oligonucleotide comprises a chimera of modified arabinose and 2'-deoxy sugars. Such an oligonucleotide has a general backbone composition of "[FANA WI NG]-[DNA GAP]-[FANA W1 NG]", or 5'RO (FANA-p)x-(DNA-p)y- (FANA-p)z-(FANA)3'OH, and more precisely has the general structure:

wherein, x>1 , y>1 , and z>0, and R is selected from the group consisting of hydrogen, thiophosphate, and a linker moiety that enhances cellular uptake of such oligonucleotide.

In another embodiment of the present invention, an antisense oligonucleotide or a therapeutic RNA has the formula:

wherein x>1 , y>1 , and z>0; R is selected from the group consisting of hydrogen, thiophosphate, and a linker moiety that enhances cellular uptake of such oligonucleotide; B is selected from the group consisting of adenine, guanine, uracil, thymine, cytosine, inosine, and 5-methylcytosine; Y at the internucleotide phosphate linkage is selected from the group consisting of sulfur, oxygen, methyl, amino, alkylamino, dialkylamino (the alkyl group having one to about 20 carbon atoms), methoxy, and ethoxy; X at the furanose ring (position 4') is selected from the groups oxygen, sulfur, and methylene (CH₂); and Z at the 2' position of the sugar ring is selected from the group consisting of a halogen (fluorine, chlorine, bromine, iodine), alkyl, alkylhalide (e. g.,- CH₂F), allyl, amino, aryl, alkoxy, and azido.

In another embodiment of the present invention, a therapeutic RNA or an antisense oligonucleotide has the formula:

wherein x>1 , y>1 , and z>0; R is selected from the group consisting of hydrogen, thiophosphate, and a linker moiety that enhances cellular uptake of such oligonucleotide; B is selected from the group consisting of adenine, guanine, uracil, thymine, cytosine, inosine, and 5-methylcytosine. In accordance with another embodiment of the present invention there is provided an antisense oligonucleotide or a therapeutic RNA targeting DRR which has the formula:

wherein x>1 , y>1 , and z>0; R is selected from the group consisting of hydrogen, thiophosphate, and a linker moiety that enhances cellular uptake of such oligonucleotide; B is selected from the group consisting of adenine, guanine, uracil, thymine, cytosine, inosine, and 5-methylcytosine; Y at the internucleotide phosphate linkage is selected from the group consisting of sulfur, oxygen, methyl, amino, alkylamino, dialkylamino (the alkyl group having one to about 20 carbon atoms), methoxy, and ethoxy; X at the furanose ring (position 4') is selected from the groups oxygen, sulfur, and methylene (CH₂) ; and Z at the 2' position of the sugar ring is selected from the group consisting of a halogen (fluorine, chlorine, bromine, iodine), hydroxyl, alkyl, alkylhalide (e. g.,-CH₂F), allyl, amino, aryl, alkoxy, and azido.

In other embodiments, antisense oligonucleotides entirely made up of FANA units, as described in WO/1999/67378 are used in the compositions and methods of the invention. For example, an antisense oligonucleotide may comprise sugar-modified oligomers composed of P- D- arabinonucleotides (i. e., ANA oligomers) and 2'- deoxy-2'-fluoro-B-D- arabinonucleosides (i. e., 2'F-ANA oligomers), such as those described in International PCT publication no. WO/1999/67378.

In still other embodiments, an antisense oligonucleotide of the invention may be a nucleic acid ligand (or "aptamer") capable of forming a G- tetrad and comprising at least one arabinose modified nucleotide. For example, an arabinose modified nucleotide may be 2' -deoxy-2' - fluoroarabinonucleotide (FANA). An arabinose modified nucleotide may be in the loop of the G-Tetrad or alternatively a guanosine residue of the G-tetrad.

In an embodiment, an aptamer is fully substituted with arabinonucleotides. For example: 5' -AAAAAAAAAAAAAAA-3' . In another embodiment, an antisense RNA is a chimera constructed from 2' - deoxyribonucleotide (DNA) and 2'-deoxy-2' -fluoroarabinonucleotide (FANA). In other embodiments of the invention, an antisense RNA of the invention is an aptamer having a sugar-phosphate backbone composition selected from any combination of arabinose and deoxyribose nucleotides. In a particular embodiment, arabinose nucleotides are 2'-deoxy-2' -fluoroarabinonucleotide (FANA). In other embodiments of the invention, arabinonucleotide comprises a 2' substituent selected from the group consisting of fluorine, hydroxyl, amino, azido, alkyl, alkoxy, and alkoxyalkyi groups. In a further embodiment of the invention, an alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and functionalized alkyl groups such as ethylamino, propylamino and butylamino groups. In embodiments, an alkoxy group is selected from the group consisting of methoxy, ethoxy, proproxy and functionalized alkoxy groups such as -0(CH₂)_q-R, where q=2-4 and -R is a - NH₂, -OCH₃, or -OCH₂CH₃ group. In embodiments, an alkoxyalkyl group is selected from the group consisting of methoxyethyl , and ethoxyethyl. In embodiments, a 2' substituent is fluorine and the arabinonucleotide is a 2'- fluoroarabinonucleotide (FANA). In one embodiment, a FANA nucleotide is araF-G and araF-T.

In other embodiments of the invention, an antisense oligonucleotide of the invention is an aptamer comprising one or more internucleotide linkages selected from the group consisting of: a) phosphodiester; b) phosphotriester; c) phosphorothioate;d) methylphosphonate; e) boranophosphate; and f) any combination of (a) to (e) .

In yet other embodiments, antisense oligonucleotides such as those described in PCT international publication no. WO/2007/038869 are used in the compositions and methods of the invention. Such oligonucleotides may be nucleic acid ligands (or aptamers) capable of forming a G- tetrad and comprising at least one arabinose modified nucleotide. In an embodiment, an arabinose modified nucleotide is 2' -deoxy-2' -fluoroarabinonucleotide (FANA). An arabinose modified nucleotide is preferably in the loop of the G- Tetrad or alternatively a guanosine residue of the G-tetrad.

In an embodiment, an aptamer may have any number of arabinonucleotides at any location in the aptamer, for example:

5 ' -ADADADADADADADA-3 ' ; 5 ' -AADADDADDDAADAD-3 ' ;

5 ' -AAAADAAADADDDAD-3 '; etc,

wherein A is an arabinonucleotide and D is a 2'- deoxyribonucleotide .

In other embodiments of the invention, an aptamer is fully substituted with arabinonucleotides. For example: 5' -AAAAAAAAAAAAAAA-3 ' .

In other embodiments of the present invention, chimeras constructed from 2' -deoxyribonucleotide (DNA) and 2'- deoxy-2' -fluoroarabinonucleotide (FANA) capable of binding DRR selectively are provided.

In other embodiments, an antisense RNA of the invention is an aptamer of any one of sequence 5'-GGTTGGTGTGGTTGG-S', dT₂G₄T₂ and d [G₄T₄G₄Jn, having a sugar-phosphate backbone composition selected from any combination of arabinose and deoxyribose nucleotides. Arabinose nucleotides may be 2'- deoxy-2' -fluoroarabinonucleotide (FANA).

In other embodiments of the invention, an arabinonucleotide comprises a 2' substituent selected from the group consisting of fluorine, hydroxyl, amino, azido, alkyl, alkoxy, and alkoxyalkyi groups. In a further embodiment of the invention, an alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and functionalized alkyl groups such as ethylamino, propylamino and butylamino groups. In embodiments, an alkoxy group is selected from the group consisting of methoxy, ethoxy, proproxy and functionalized alkoxy groups such as -0(CH₂)_q-R, where q=2-4 and -R is a -NH₂, -OCH₃, or -OCH₂CH₃ group. In embodiments, an alkoxyalkyi group is selected from the group consisting of methoxyethyl , and ethoxyethyl . In embodiments, a 2' substituent is fluorine and the arabinonucleotide is a 2'- fluoroarabinonucleotide (FANA). In an embodiment, a FANA nucleotide is araF-G and araF-T.

In other embodiments of the invention, an antisense RNA is an aptamer comprising one or more internucleotide linkages selected from the group consisting of: a) phosphodiester; b) phosphotriester; c) phosphorothioate; d) methylphosphonate; e) boranophosphate; and f) any combination of (a) to (e).

In another embodiment, an antisense RNA is an aptamer with at least one nucleotide of the aptamer, preferably in a loop of the aptamer that forms a G-tetrad, replaced with an arabinose modified nucleotide, preferably 2'- deoxy-2'- fluoroarabinonucleotide (FANA).

In other embodiments, antisense oligonucleotides such as those described in WO/2003/037909 may be used in the methods and compositions of the invention. In brief, such oligonucleotides have the structure: [R'-XJ_a-R²]a

wherein a is greater than or equal to 1 ; wherein each of R¹ and R² are independently at least one nucleotide; and wherein X is an acyclic linker. In an embodiment, an oligonucleotide comprises at least one modified deoxyribonucleotide, i.e. either R¹, R² or both may comprise at least one modified deoxyribonucleotide. In an embodiment, a modified deoxyribonucleotide is selected from the group consisting of ANA, PS-ANA, PS-DNA, RNA-DNA and DNA-RNA chimeras, PS- [RNA-DNA] and PS-[DNA- RNA] chimeras, PS- [ANA-DNA] and PS-[DNA-ANA] chimeras, RNA, PS- RNA, PDE- or PS-RNA analogues, locked nucleic acids (LNA) , phosphorodiamidate morpholino nucleic acids, N3'-P5' phosphoramidate DNA, cyclohexene nucleic acid, alpha-L-LNA, boranophosphate DNA, methylphosphonate DNA, and combinations thereof. In an embodiment, an ANA is FANA (e.g. PDE- or PS-FANA).

As used herein, "PS" refers to a phosphorothioate linkage. For example, "PS-DNA" refers to DNA with phosphorothioate linkages between nucleotides. PS-DNA is known to induce RNase H degradation of targeted RNAs and is resistant to degradation by serum and cellular nucleases. "PDE" refers to a phosphodiester linkage.

In an embodiment, the above-mentioned PDE- or PS-RNA analogues are selected from the group consisting of 2' -modified RNA wherein the 2'- substituent is selected from the group consisting of alkyl, alkoxy, alkylalkoxy, F and combinations thereof. In an embodiment, an acyclic linker is selected from the group consisting of an acyclic nucleoside and a non-nucleotidic linker. In embodiments, an acyclic nucleoside is selected from the group consisting of purine and pyrimidine seconucleosides. In embodiments, a purine seconucleoside is selected from the group consisting of secoadenosine and secoguanosine. In embodiments, a pyrimidine seconucleoside is selected from the group consisting of secothymidine, secocytidine and secouridine.

In an embodiment, a non-nucleotidic linker comprises a linker selected from the group consisting of an amino acid and an amino acid derivative. In embodiments, an amino acid derivative is selected from the group consisting of (a) an N- (2-aminoethyl) glycine unit in which an heterocyclic base is attached via a methylene carbonyl linker (PNA monomer); and (b) an O-PNA unit. According to a further aspect of the invention, there is provided an antisense oligonucleotide chimera of general structure lb:

AO N X-—\ AO N 2 -3 ' lb wherein n is greater than or equal to 1 . With reference to structure lb above, "AON1 " is an oligonucleotide chain, which in embodiments is selected from the group consisting of ANA (e.g. FANA), DNA, PS-DNA, 5' -RNA-DNA-3' chimeras, as well as other RNase H-competent oligonucleotides, for example arabinonucleic acids (2' -OH substituted ANA) (Damha, M.J. et al . J. Am . Chem . Soc . 1998, 120, 12976), cyclohexene nucleic acids (Wang J. et al . J. Am . Chem . Soc. 2000, 122, 8595), boranophosphate linked DNA (Rait, V.K. et al . Antisense Nucleic Acid Drug Dev. 1999, 9, 53), and alpha-L- locked nucleic acids (Sorensen, M.D. et al . J. Am . Chem . Soc. 2002, 124, 2164) or combinations thereof; and "AON2" is an oligonucleotide chain, which in embodiments is selected from the group consisting of FANA, DNA, PS-DNA, 5'-DNA-RNA-3' chimeras, as well as other RNase H-competent oligonucleotides such as those described above, or combinations thereof. Internucleotide linkages of the AON1 and AON2 include but are not necessarily limited to phosphodiester, phosphotriester, phosphorothioate, methylphosphonate, and/or phosphoramidate (5'N-3'P and 5'P-3'N) groups. A substituent directly attached to the C2'-atom of the arabinose sugar in ANA-X-ANA chimera constructs includes but is not limited to fluorine, hydroxyl, amino, azido, alkyl (e.g. 2' -methyl, ethyl, propyl, butyl, etc.), and alkoxy groups (e.g., 2'-OMe, 2'-OEt, 2'-OPr, 2'-0Bu, 2'-OCH₂CH₂OMe, etc.).

In other embodiments, a therapeutic RNA or an antisense

oligonucleotide of the invention has the structure:

wherein each of m, n, q and a are independently integers greater than or equal to 1 ; wherein each of R and R² are independently at least one nucleotide, wherein each of Z¹ and Z² are independently selected from the group consisting of an oxygen atom, a sulfur atom, an amino group and an alkylamino group;

wherein each of Y¹ and Y² are independently selected from the group consisting of oxygen, sulfur and NH; and wherein R³ is selected from the group consisting of H, alkyl, hydroxyalkyl, alkoxy, a purine, a pyrimidine and combinations thereof. In embodiments, R³ is adenine or guanine, or derivatives thereof. In embodiments, R³ is thymine, cytosine, 5- methylcytosine, uracil, or derivatives thereof. In embodiments, each of R¹ and R² noted above are independently selected from the group consisting of ANA, PS-ANA, PS-DNA, RNA-DNA and DNA-RNA chimeras, PS- [RNA- DNA] and PS- [DNA-RNA] chimeras, PS- [ANA-DNA] and PS-[DNA-ANA] chimeras, alpha-L-LNA, cyclohexene nucleic acids, RNA, PS-RNA, PDE- or PS-RNA analogues, locked nucleic acids (LNA) , phosphorodiamidate morpholino nucleic acids, N3'-P5' phosphoramidate DNA,

methylphosphonate DNA, and combinations thereof. In embodiments, each of R¹ and R² noted above independently may comprise at least two nucleotides connected via an internucleotide linkage, wherein said internucleotide linkage is selected from the group consisting of phosphodiester, phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate (5'N-3'P and 5'P-3'N) groups and combinations thereof. In other embodiments, each of R¹ and R² noted above independently comprise ANA. In embodiments the above-noted ANA comprises a 2 ' - substituent selected from the group consisting of fluorine, hydroxyl, amino, azido, alkyl (e.g. methyl, ethyl, propyl and butyl) and alkoxy (e.g. methoxy, ethoxy, propoxy, and methoxyethoxy) groups. In an embodiment, a 2' - substituent is fluorine and said ANA is FANA. In embodiments, an alkyl group is selected from the group consisting of methyl, ethyl, propyl and butyl groups. In embodiments, an alkoxy group is selected from the group consisting of methoxy, ethoxy, propoxy, and methoxyethoxy groups .

In other embodiments, a therapeutic RNA or an antisense

oligonucleotide of the invention targeting DRR is selected from the group consisting of:

wherein R¹, R², n, a, Z¹, Z², Y¹ and Y² are as defined above and each of R⁴ and R⁵ are independently selected from the group consisting of a purine (e.g. adenine and guanine or derivatives thereof) and a pyrimidine (e.g.

thymine,

cytosine, uracil, or derivatives thereof) . In an embodiment, R¹ and R² are PDE-FANA; and a=l. In an embodiment, R¹ and R² are PS-FANA; and a=l. In an embodiment, R¹ is [FANA-DNA] ; R² is [DNA-FANA] ; and a=l. In an embodiment, R¹ is [ FANA-DNA] ; R² is FANA; and a=l. In an embodiment, R¹ is FANA; R² is [DNA-FANA] ; and a=l. In an embodiment, R¹ and R² are PS- DNA; and a=\. In an embodiment, R¹ is PDE- [RNA-DNA] , R² is PDE- [DNA- RNA] ; and a=l. In an embodiment, R¹ is RNA; R² is [DNA-RNA]; and a=l. In an embodiment, R¹ is S- [ (2'0-alkyl) RNA-DNA] ; R² is S-[DNA- (20- alkyl)RNA] ; and a=l. In an embodiment, R¹ is S- [ (2'0-alkyl) RNA-DNA] ; R² is S-[ (2'0-alkyl) RNA] ; and a=l. In an embodiment, R¹ is S- [ (2'0-alkyl) RNA] ; R² is S-[DNA- (2'0-alkyl) RNA] ; and a=l. In an embodiment, R¹ is S- [ (2' O- alkoxyalkyl) RNA-DNA] ; R² is S- [DNA- (2' O-alkoxyalkyl) RNA] ; and a=l. In an embodiment, R¹ is S- [ (2' O-alkoxyalkyl) RNA-DNA] ; R² is S-[ (2 O- alkoxyalkyl) RNA] ; and a=l . In an embodiment, R¹ is S- [ (2' O-alkoxyalkyl) RNA] ; R² is S- [DNA- (2' O-alkoxyalkyl) RNA] ; and a=l. In an embodiment, R¹ is PDE- [ (2' O-alkyl-RNA) -DNA] ; R² is PDE- [DNA- (2'0-alkyl RNA)]; and a=l. In an embodiment, R¹ is PS- [ (2' O-alkyl-RNA) -DNA] ; R² is PS- [DNA- (2'0-alkyl RNA)]; and a=l. In an embodiment, R¹ is PDE- [ (2' O-alkoxyalkyl- RNA) -DNA] ; R² is PDE- [DNA- (2' O-alkoxyalkyl RNA)]; and a=l.

In an embodiment, R¹ is PS- [ (2' O-alkoxyalkyl-RNA) -DNA] ; R² is PS- [DNA- (2' O-alkoxyalkyl RNA)]; and a=l. In an embodiment, R¹ and R² are PDE- [FANA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n=4. In an embodiment, R¹ is PS- [FANA]; R² is PDE- [FANA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n=4. In an embodiment, R¹ is FANA; R² is PS-FANA; a=l; and the

oligonucleotide has structure lib in which Y¹, Y², Z¹ and Z² are oxygen and n=4 In an embodiment, R¹ and R² are PS- [FANA]; a=l; and the

oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n=4. In an embodiment, R¹ is PS-[DNA]; R² is PDE- [DNA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n=4. In an embodiment, R¹ is PDE- [DNA]; R² is PS- [DNA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n= . In an embodiment, R¹ and R² are PS- [DNA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z2 are both oxygen or sulfur, and n=4. In an embodiment, R¹ and R² are PDE- [FANA]; a=l; and the

oligonucleotide has structure lie in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur. In an embodiment, R¹ is PS- [FANA]; R² is PDE- [FANA]; a=l; and the oligonucleotide has structure lie in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur. In an embodiment, R¹ is PDE- [FANA]; R² is PS- [FANA]; a=l; and the oligonucleotide has structure lib in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur, and n=4.

In an embodiment, R¹ and R² are PS- [FANA]; a=l; and the oligonucleotide has structure lie in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur. In an embodiment, R¹ is PS- [DNA]; R² is PDE- [DNA]; a=l; and the oligonucleotide has structure lie in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur. In an embodiment, R¹ is DNA; R² is PS-DNA; a=l; and the oligonucleotide has structure He. In an embodiment, R¹ and R² are PS- [DNA]; a=l; and the oligonucleotide has structure lie in which Y¹, Y² are oxygen; Z¹ , Z² are both oxygen or sulfur. In an embodiment, a=2 and each of R¹ and R² independently consist of at least 3 nucleotides, in a further embodiment, of 3- 8 nucleotides. In an embodiment, a=3 and each of R¹ and R² independently consist of at least 2 nucleotides, in a further embodiment, wherein each of R¹ and R² independently consist of 2-6 nucleotides. In an embodiment, the oligonucleotide is antisense to a target RNA.

"RNA interference" or "RNAi" is a term initially applied to a phenomenon observed in plants and worms where double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo. RNAi involves using small interfering RNA, or siRNA, to target an mRNA sequence. With siRNA, a cell utilizes a protein complex called RISC to destroy the mRNA, thereby preventing the production of a disease-causing protein.

In the present context, the term "therapeutic RNA" relates to oligonucleotides and similar species, for use in reducing or inhibiting DRR expression. Non-limiting examples of therapeutic RNAs include antisense RNAs, RNAi, siRNA, dsRNA, shRNA, and other like RNAs, as are known in the art to reduce expression of a target RNA; in an embodiment, a target RNA is DRR mRNA or a fragment or portion thereof.

Many different types of therapeutic RNAs are known in the art. Other examples of therapeutic RNAs which are encompassed by the invention include: RNA interfering (RNAi) agents that perform gene knockdown of message (mRNA) by degradation or translational arrest of the mRNA, e.g., inhibition of tRNA and rRNA functions; small interfering RNA (siRNA); short hairpin RNA (shRNA); microRNA, non-coding RNA and the like; Short RNAs; Dicer-substrate siRNAs (DsiRNAs); UsiRNAs; Self-delivering RNA (sdRNA); siNA; nucleotide based agents inhibiting the pre-mRNA maturation step of polyA tail addition; Ul adaptors; aptamers; triple-helix formation; DNAzymes; antisense; Morpholinos (e.g., PMO, phosphorodiamidate morpholino oligo); ribozymes; and combinations thereof.

In some embodiments, a therapeutic RNA encompasses oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding DRR or a portion or fragment thereof. In other embodiments, oligonucleotides comprising the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, are encompassed.

In an embodiment, a therapeutic RNA of the invention is an oligonucleotide which is complementary to or specifically hybridizes with a fragment or portion of the DRR mRNA. Non-limiting examples of a fragment or portion of DRR mRNA to which a therapeutic RNA is complementary or specifically hybridizes include the following: nucleotides 170-190 of the DRR mRNA; nucleotides 175-195 of the DRR mRNA; nucleotides 180-200 of the DRR mRNA; nucleotides 185-205 of the DRR mRNA; nucleotides 190-210 of the DRR mRNA; nucleotides 195-215 of the DRR mRNA; nucleotides 200- 220 of the DRR mRNA; nucleotides 205-225 of the DRR mRNA; nucleotides 210-230 of the DRR mRNA; nucleotides 215-235 of the DRR mRNA; nucleotides 220-240 of the DRR mRNA; nucleotides 225-245 of the DRR mRNA; nucleotides 230-250 of the DRR mRNA; nucleotides 235-255 of the DRR mRNA; nucleotides 240-260 of the DRR mRNA; nucleotides 245-265 of the DRR mRNA; nucleotides 250-270 of the DRR mRNA; nucleotides 255- 275 of the DRR mRNA; nucleotides 260-280 of the DRR mRNA; nucleotides 265-285 of the DRR mRNA; nucleotides 270-290 of the DRR mRNA; nucleotides 275-295 of the DRR mRNA;nucleotides 280-300 of the DRR mRNA; nucleotides 285-305 of the DRR mRNA; nucleotides 290-310 of the DRR mRNA; nucleotides 295-315 of the DRR mRNA; nucleotides 300-320 of the DRR mRNA; nucleotides 305-325 of the DRR mRNA; nucleotides 310- 330 of the DRR mRNA; nucleotides 315-335 of the DRR mRNA; nucleotides 320-340 of the DRR mRNA; nucleotides 325-345 of the DRR mRNA; nucleotides 330-350 of the DRR mRNA; nucleotides 335-355 of the DRR mRNA; nucleotides 340-360 of the DRR mRNA; nucleotides 345-365 of the DRR mRNA; nucleotides 350-370 of the DRR mRNA; nucleotides 355-375 of the DRR mRNA; nucleotides 360-380 of the DRR mRNA; nucleotides 365- 385 of the DRR mRNA; nucleotides 370-390 of the DRR mRNA; nucleotides 375-395 of the DRR mRNA; nucleotides 380-400 of the DRR mRNA; or a fragment, portion or derivative thereof.

In other embodiments, a therapeutic RNA has a sequence complementary to or specifically hybridizing to nucleotides 425 to 439 of the DRR mRNA, or complementary to or specifically hybrizing to nucleotides 420 to 444 of the DRR mRNA, or complementary to or specifically hybrizing to nucleotides 415 to 439 of the DRR mRNA, or complementary to or specifically hybrizing to nucleotides 424 to 439 of the DRR mRNA, 423 to 439, 422 to 439, 421 to 439, or 420 to 439, or 420 to 434 of the DRR mRNA; or a fragment, portion or derivative thereof.

It shall be understood that nucleic acids hybridizing to an additional 1 to 3 nucleotides at either end or to a smaller fragment or to a derivative of the recited sequences and regions are also encompassed. In addition, nucleic acid sequences may include extra nucleotides required for function of a therapeutic RNA molecule, such as those required to form a short hairpin loop.

In yet other embodiments, a therapeutic RNA of the invention is an antisense oligonucleotide which has the structure of an altimer, a gapmer, an aptamer, and/or comprises one or more modified nucleotide such as 2' - deoxy-2' -fluoroarabinonucleotide (FANA), as described herein.

DNA molecules encoding a therapeutic RNA of the invention and expression vectors suitable for production of therapeutic RNAs of the invention are also provided. Therapeutic RNAs are also referred to as "RNA tools" herein.

In the present context, the expression "dsRNA" relates to double stranded RNA capable of causing RNA interference. In accordance with the present invention, any suitable double-stranded RNA fragment capable of directing RNAi or RNA-mediated gene silencing of the target gene can be used.

As used herein, a "double-stranded ribonucleic acid molecule (dsRNA)" refers to any RNA molecule, fragment or segment containing two strands forming an RNA duplex, notwithstanding the presence of single stranded overhangs of unpaired nucleotides. A double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to the target nucleotide sequence (i.e. to at least a portion of the mRNA transcript) of the target gene to be down- regulated. The other strand of the double-stranded RNA is complementary to the target nucleotide sequence.

A double-stranded RNA need only be sufficiently similar to a mRNA sequence of the target gene to be down-regulated such that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and a nucleotide sequence of a dsRNA sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs.

According to the invention, a "dsRNA" or "double stranded RNA", whenever said expression relates to RNA that is capable of causing interference, may be formed from two separate (sense and antisense) RNA strands that are annealed together. An antisense (or "guide") strand is a strand that is complementary to the mRNA, whereas a sense (or "passenger") strand of a siRNA duplex has a sequence that is complementary to the guide or antisense strand (and identical to a region of an mRNA strand).

Alternatively, a dsRNA may have a foldback stem-loop or hairpin structure wherein the two annealed strands of the dsRNA are covalently linked. In this embodiment, sense and antisense strands of a dsRNA are formed from different regions of a single RNA sequence that is partially self- complementary.

As used herein, the term "RNAi molecule" is a generic term referring to double stranded RNA molecules including small interfering RNAs (siRNAs), hairpin RNAs (shRNAs), and other RNA molecules which can be cleaved in vivo to form siRNAs. RNAi molecules can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical or substantially identical to only a region of the target nucleic acid sequence.

The subject RNAi molecules can be "small interfering RNAs" or "siRNAs." siRNA molecules are usually synthesized as double stranded molecules in which each strand is around 19-32 nucleotides in length, or around 21 -31 nucleotides in length, or around 21 to 23 nucleotides in length, or around 23 to 29 nucleotides in length, or around 29 nucleotides in length. siRNAs are understood to recruit nuclease complexes and guide the complexes to a target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, siRNA molecules comprise a 3' hydroxyl group. In certain embodiments, siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence of the enzyme dicer.

Alternatively, a RNAi molecule is in the form of a hairpin structure, named as hairpin RNA or shRNA. Hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.

RNAi molecules may include modifications to either the phosphate- sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.

In some cases, at least one strand of an RNAi molecule has a 3' overhang from about 1 to about 6 nucleotides in length, and for instance from 2 to 4 nucleotides in length. More preferably, 3' overhangs are 1 -3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is blunt-ended or also has an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance stability of the RNAi molecules, 3' overhangs can be stabilized against degradation. In one embodiment, an RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi.

Production of RNAi molecules can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. RNAi molecules may be produced enzymatically or by partial/total organic synthesis. Any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.

RNAi molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify RNAi molecules. Alternatively, non-denaturing methods, such as non- denaturing column chromatography, can be used to purify RNAi molecules. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, and/or affinity purification with antibody can be used to purify RNAi molecules. Nucleic Acids, RNAi Molecules and Expression Constructs

The invention is in one aspect related to use of a nucleic acid sequence (e.g. a therapeutic RNA, a DNA encoding same, or a vector producing same) to prepare an antisense RNA or RNAi molecule suitable for reducing expression of a target gene, e.g. DRR, in tumor cells, e.g. glioma cells.

As used herein the term "reducing the expression of a target gene" refers to the ability of a present therapeutic RNA, e.g. antisense, RNAi or other therapeutic molecules, to block expression of the target gene in a specific and post-transcriptional manner.

In one embodiment the invention relates to the use of an RNA sequence to prepare a therapeutic RNA molecule as defined herein. In an embodiment, a RNA molecule is an RNAi molecule, such as a siRNA molecule. Said therapeutic RNA molecule is characterized by one or more, and in one embodiment by all, of the following criteria: having at least 50% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity with the target mRNA; having a sequence which targets the exon area of the target gene; and/or showing a preference for targeting the 3' end of the target gene rather than for targeting the 5' end of the target gene. In some embodiments, a target gene is DRR or a fragment of the gene encoding DRR.

In a further embodiment, a therapeutic RNA molecule may be further characterized by one or more, or by all, of the following criteria: having a nucleic acid length of between 15 to 25 nucleotides, or of between 18 to 22 nucleotides, or of 19 nucleotides, or of between 19 to 33 nucleotides, 21 to 31 nucleotides, or of 29 nucleotides; or of 13 to 17 nucleotides, having a GC content comprised between 30 and 50%; showing a TT(T) sequence at its 3' end; showing no secondary structure when adopting the duplex form; having a Tm (melting temperature) of lower than 20° C; or having the nucleotides indicated in SEQ ID NOs: 1 , 2, 5 -10, 14-16, 17/18, 19/20, 21 /22 or 23/24 (nucleotide sequences are given in Table 1 ; "nt" stands for nucleotide). In an embodiment, a therapeutic RNA molecule has a nucleic acid length of 15 nucleotides.

In another embodiment, a therapeutic RNA comprises 15 nucleotides complementary to DRR mRNA with additional nucleotides necessary to improve function as a therapeutic RNA, such as sequences which facilitate creation of a short hairpin loop in the therapeutic nucleic acid or RNA. In yet another embodiment, a therapeutic nucleic acid molecule has the sequence of SEQ ID NO: 1 or 2. In another embodiment, a therapeutic nucleic acid molecule has a sequence complementary to or specifically hybrizing to nucleotides 425 to 439 of the DRR mRNA, or complementary to or specifically hybrizing to nucleotides 420 to 444 of the DRR mRNA or a fragment or derivative thereof, or complementary to or specifically hybrizing to nucleotides 415 to 439 of the DRR mRNA or a fragment or derivative thereof, or complementary to or specifically hybrizing to nucleotides 424 to 439 of the DRR mRNA, 423 to 439, 422 to 439, 421 to 439, or 420 to 439, or 420 to 434 of the DRR mRNA. It shall be understood that nucleic acids hybridizing to an additional 1 to 3 nucleotides at either end or to a smaller fragment or derivative of the recited sequences are also encompassed. In addition, nucleic acid sequences may include extra nucleotides required for function of the therapeutic RNA molecule, such as those required to form a short hairpin loop.

In an embodiment, a therapeutic RNA of the invention comprises the sequences provided herein, for example SEQ ID NOs: 1 , 2, 5 - 10, 14-16, 17/18, 19/20, 21 /22 or 23/24. In another embodiment, a therapeutic RNA of the invention consists of the sequences provided herein, for example SEQ ID NOs: 1 , 2, 5 - 10, 14-16, 17/18, 19/20, 21 /22 or 23/24.

In another embodiment, a therapeutic RNA of the invention has the sequence of SEQ ID NO: 14, 15 or 16. In yet another embodiment, a therapeutic RNA of the invention has the sequence of SEQ ID NO: 17/18, 19/20, 21 /22 or 23/24.

TABLE 1. Exemplary nucleic acid sequences.

sequence

b For siRNA sequences, "p" means 5' phosphate; uppercase indicates RNA and lowercase indicates DNA; "S" indicates sense strand; and "AS" indicated antisense strand.

c Nucleotides which are italicized and double underlined are FRNA (also referred to as 2'F-RNA). It will be understood by those in the art that siRNAs are generally duplexes of two strands, a sense strand and an antisense strand; both strands are listed in Table 1 .

The siRNA duplex targeting DRR referred to herein as "siRNAI " is a duplex of SEQ ID NOs: 17 and 18, where SEQ ID NO: 17 is the sense strand and SEQ ID NO: 18 is the antisense strand. The siRNAI duplex is also referred to herein as "SEQ ID NO: 17/18", and "DRR1 siRNA".

Similarly, "siRNA2" is a duplex of SEQ ID NOs: 19 and 20, where SEQ ID NO: 19 is the sense strand and SEQ ID NO: 20 is the antisense strand. The siRNA2 duplex is also referred to herein as "SEQ ID NO: 19/20" and "DRR2 siRNA".

"siRNA2-Cy5" is a duplex of SEQ ID NOs: 21 and 22, where SEQ ID NO: 21 is the sense strand and SEQ ID NO: 22 is the antisense strand. The siRNA2-Cy5 duplex is also referred to herein as "SEQ ID NO: 21/22".

"siRNA3" is a duplex of SEQ ID NOs: 23 and 24, where SEQ ID NO:

23 is the sense strand and SEQ ID NO: 24 is the antisense strand. The siRNA3 duplex is also referred to herein as "SEQ ID NO: 23/24" and "DRR3 siRNA".

The "siRNA control sequence" in Table 1 is an siRNA which does not target DRR. The siRNA control sequence is a duplex of SEQ I D NOs: 25 and 25, where SEQ ID NO: 25 is the sense strand and SEQ ID NO: 26 is the antisense strand. The siRNA control sequence duplex is also referred to herein as "SEQ ID NO: 25/26" and "DRR4 siRNA" and "siRNA4".

Effective antisense sequences targeting DRR were designed using an antisense oligonucleotide (AON) sequence selection tool available from Integrated DNA Technologies (IDT®)

(http://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx7sour ce=menu). Predicted antisense sequences were checked using BLAST alignment tools (NCBI) to check for absence of off-target hits. Additionally, mRNA secondary structure of a section of the DRR mRNA was predicted using the MFOLD tool (http://mfold.rna. albany.edu/?q=mfold/RNA-Folding- Form) to look for mRNA-accessibility for AON binding. AONs targeting DRR are shown in Table 1 (SEQ ID NOs: 14, 15 and 16), and control AONs not targeting DRR were also prepared for control experiments (SEQ ID NOs: 1 1 , 12 and 13).

In another embodiment, the invention is related to the use of an RNA sequence containing any of the following sequences: SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, to prepare a therapeutic RNA molecule, for example an antisense or RNAi molecule, suitable for reducing expression of DRR in glioma cells.

In the context of the present invention, the terms "fragment" and

"derivative" refer to nucleic acids that may differ from the original nucleic acid in that they are extended or shortened on either the 5' or the 3' end, on both ends or internally, or extended on one end, and shortened on the other end, provided that the function of the resulting molecule, namely down-regulation of a target gene, is not abolished or inhibited. The terms "fragment" and "derivative" also refer to nucleic acids that may differ from an original nucleic acid in that one or more nucleotides of the original sequence are substituted by other nucleotides and/or (chemically) modified by methods available to a skilled person, provided that function of the resulting molecule is not abolished or inhibited. The "fragment" and "derivative" may typically show at least 80%, e.g., at least 85%, at least 90%, at least 95% or even at least 99% sequence identity to the original nucleic acid. Sequence identity between two nucleotide sequences can be calculated by aligning the said sequences and determining the number of positions in the alignment at which the two sequences contain the same nucleic acid base vs. the total number of positions in the alignment.

It should be understood that fragments and derivatives of nucleic acid sequences in Table 1 which retain an ability to reduce or decrease DRR expression are encompassed. For example, nucleic acid sequences comprising about 12, about 13, about 14, about 15, about 16, about 17, about 18 or about 19 contiguous nucleotides from sequences given in Table 1 , and retaining an ability to reduce/decrease DRR expression, are encompassed.

It shall be clear to a person of skill in the art that any of the above- given sequences or complementary sequences thereof may be used to prepare a therapeutic RNA molecule, e.g. an antisense or RNAi molecule, for example a double stranded RNA molecule. A person of skill in the art knows how to prepare an antisense or RNAi molecule when the above disclosed nucleic acids, particularly RNAs, are provided. Briefly, required nucleic acids may be synthesized by any available method and strands annealed, as required, under appropriate conditions. Annealing conditions, e.g. temperatures and incubation periods, may be adjusted according to the respective nucleic acid sequences.

In a particular embodiment the invention relates to the use of an RNA sequence containing the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, a fragment or derivative thereof, to prepare a therapeutic RNA molecule, such as an RNAi molecule, and preferably an siRNA molecule.

In another embodiment, the invention relates to the use of an RNA sequence containing the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, to prepare an antisense molecule.

In order to exert a desired function, e.g., reducing expression of DRR in glioma cells, therapeutic RNAs (antisense, RNAi molecules, siRNA and so on) according to the invention are prepared from ribonucleic acids of the present invention as defined above, are delivered into target cells, e.g., human GBM cells.

There are several well-known methods of introducing (ribo)nucleic acids into animal cells, any of which may be used in the present invention and which depend on the host. At the simplest, a nucleic acid can be directly injected into a target cell/target tissue. Other methods include fusion of a recipient cell with bacterial protoplasts containing a nucleic acid, use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors, electroporation, and the like. Other techniques or methods which are suitable for delivering therapeutic RNA molecules as defined herein to target cells include continuous delivery of an RNAi molecule as defined herein from poly(lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) RNAi molecule(s) into micropumps delivering the product in the cavity of surgical resection to tumor cells still present at a site of surgery, e.g., in a hole of neurosurgical resection to tumor cells still present in the brain parenchyma, as has been detailed previously for the use of other anti-migratory compounds (see for example Lefranc et al., Neurosurgery 52: 881 -891 , 2003). Convection- enhanced delivery (as detailed by Kawakami et al., J Neurosurg 101 : 1004- 101 1 , 2004) of stabilized molecules, e.g., RNAi molecules as defined herein, can also be used. Another possibility is use of implantable drug-releasing biodegradable microspheres, as those reviewed by Menei and Benoit, Acta Neurochir 88:51 -55, 2003. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used. Another approach is to use either an Ommaya reservoir (micropumps) delivering RNA molecules versus encapsulated RNA molecules in biodegradable microspheres, or both approaches at the same time.

The main obstacle to achieve in vivo gene silencing with therapeutic RNAs, such as antisense and RNAi technologies, is delivery. To improve thermal stability, resistance to nuclease digestion and to enhance cellular uptake of the RNAs, various approaches have been tested in the art. These include chemical modifications like locked nucleic acid, phosphorothioate substitution, 2'-fluoro substitution, 2'-0-methyl substitution, stabilized stealth™ RNAi (Invitrogen), encapsulation of RNA tools in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticules or dendrimers, poly(lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, co-injection of the RNAi tools with a protective agent, and so on. It shall be understood that these methods and others known in the art may be used in the methods of the present invention.

In one aspect, RNA tools of the present invention, optionally stabilized, encapsulated or otherwise modified as above, are delivered at a site of a tumor, e.g., a primary tumor and/or metastases. A manner of achieving localized delivery is use of an Ommaya reservoir as described elsewhere. Another way of targeting present RNA tools to tumor cells is to use antibody- directed, cell type-specific delivery. For example, RNAi (e.g., siRNA) can be complexed with Fab specifically recognizing tumor cells, such as Fab- protamine-complexed (Song et al., Nat Biotechnol 23:709-717, 2005), or RNAi may be encapsulated in immunoliposomes. Such antibody-targeted RNAi tools, e.g. , in the form of nanoparticles, can be administrated by various means, such as systemic administration (i.v. injection, subcutaneous injection, intramuscular injection, oral administration, nasal inhalation, etc.) or locally, e.g., using an Ommaya reservoir. In particular, convection delivery with injection at a remote date or at time of surgery may be used. Inhalative administration of the present RNA tools, e.g., in the form of nasal sprays or aerosol mixtures, may also be employed. Another option is use of nanotechnology for delivery.

In vivo delivery of RNA tools has been described, e.g., intravenous, intracerebroventricular or intranasal administration of naked or lipid- encapsulated siRNA molecules. Intravenous administration of shRNA vectors encapsulated in immunoliposomes or in viral particles have also been described and are known in the art.

The effect of a RNAi molecule, i.e. reduction of expression of a target gene, is considered to be only transient when molecules are directly applied to cells as for instance described above. In order to achieve a stable production of RNAi molecules in tumor cells it can be advantageous if a nucleic acid, preferably a DNA, encoding a respective target RNA molecule is integrated in an expression vector. Providing suitable elements, as described hereinafter, a DNA is transcribed into the corresponding RNA which is capable of forming the desired antisense or RNAi molecule. Thus, according to a further aspect of the present invention, expression constructs are provided to facilitate introduction into a host cell and/or facilitate expression and/or facilitate maintenance of a nucleotide sequence encoding therapeutic RNA molecules according to the invention. Expression constructs may be inserted into a plasmid, a virus, or a vector, which may be commercially available. In another embodiment, the invention therefore relates to the use of a DNA sequence to prepare an RNA molecule as defined herein. For example, DNA sequences may comprise DNA sequences which correspond to or encode RNA sequences depicted in SEQ ID NOs: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17/18, 19/20, 21/22 or 23/24, a linker, and a sequence complementary to the DNA. A linker is preferably 4 to 15 nucleotides in length, more preferably a linker is 4 to 10 nucleotides long and most preferably it is 4 to 8 nucleotides long. A linker can consist of any suitable nucleotide sequence. In one embodiment, DNA sequences consist of 15 nt sequences derived from the DRR gene which are separated by a 4 to 15 nucleotide linker, from the reverse complement of the same 15 nt sequences and showing an tt(t) sequence at its 3' end. In another embodiment, DNA sequences are inserted into an expression vector suitable for use in the methods provided herein. It is also contemplated in the present invention that expression of two complementary strands giving rise to a dsRNA is driven from two promoters, either the same or different. In this case, a nucleotide linker separating the two complementary strands would be omissible. It is further obvious to the one skilled in the art that in this case DNAs coding for two complementary siRNA strands can be present on one or on two expression vectors.

Expression vectors, capable of giving rise to transcripts which form dsRNA as defined herein, can for instance be cloning vectors, binary vectors or integrating vectors. The invention thus also relates to a vector comprising any of the DNA sequences described herein. The expression vector is preferably a eukaryotic expression vector, or a retroviral vector, a plasmid, bacteriophage, or any other vector typically used in the biotechnology field. Such vectors are known to a person skilled in the art. If necessary or desired, a DNA nucleic acid can be operatively linked to regulatory elements which direct synthesis of mRNA in eukaryotic cells.

To drive expression of dsRNA these vectors usually contain an RNA Polymerase I, an RNA Polymerase II, an RNA Polymerase III, T7 RNA polymerase or SP6 RNA polymerase and preferably RNA polymerase III promoters, such as the H1 or U6 promoter, since RNA polymerase III expresses relatively large amounts of small RNAs in mammalian cells and terminates transcription upon incorporating a string of 3-6 uridines. Type III promoters lie completely upstream of the sequence being transcribed which eliminates any need to include promoter sequence in the therapeutic RNA molecule. If a DNA encoding a desired RNAi molecule is to be transcribed from one promoter, the preferred DNA thus contains on each of its strands the desired coding region of the target gene and its reverse complementary sequence, wherein the coding and its reverse complementary sequences are separated by a nucleotide linker, allowing for the resulting transcript to fold back on itself to form a so-called stem-loop structure, and to form so-called shRNA molecules. The shRNA is transcribed from specific promoters, processed by the DICER RNAse into short double stranded RNA (siRNA) and incorporated into RISC (Dykxhoorn et al., Nat Rev Mol Cell Biol 4:457- 467, 2003) with subsequent inactivation of the targeted mRNA. Optionally, one or more transcription termination sequences may also be incorporated in the expression vector. The term "transcription termination sequence" encompasses a control sequence at the end of a transcriptional unit, which signals 3' processing and poly-adenylation of a primary transcript and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in an expression construct.

For therapeutic purposes, use of retroviral vectors has been proven to be most appropriate to deliver a desired nucleic acid into a target cell. It shall be understood that retroviral vectors or adenoviral vectors, of which many are known in the art, may also be used in the vectors, compositions and methods provided herein. It shall also be understood that expression vectors containing DNA sequences of the present invention can be introduced into a target cell by any of the delivery methods described above or otherwise known in the art. Uses, Compositions and Kits

Therapeutic RNA molecules, e.g. antisense RNA or RNAi, e.g. siRNA molecules, and/or vectors according to the present invention may be used as a medicament for treating cancer, preferably glioma, more preferably glioblastoma, or for the manufacture of a medicament for treating cancer, preferably glioma, more preferably glioblastoma. Therapeutic RNA molecules and/or vectors according to the present invention may also be used as a medicament for delaying progression of cancer, for example glioma, such as glioblastoma. The term "delaying the progression of cancer" as used herein, refers to a delay in cancer re-growth by more than 30%, or by more than 50%, or by more than 70% and/or to an increase in survival periods of affected subjects. In an embodiment, therapeutic RNA molecules and/or vectors according to the present invention may be used to inhibit brain cancer invasion, for example malignant glial cell (MGC) invasion.

In an embodiment, therapeutic RNA molecules, e.g., antisense RNA or RNAi, e.g., siRNA molecules, and/or vectors according to the present invention are used as a medicament for treating any cancer having an invasive phenotype and/or characterized by increased DRR expression. It should be understood that any cancer or tumor which is invasive or metastatic, and/or which has elevated DRR expression levels compared to non-cancerous cells, is contemplated for treatment with the therapeutic RNA molecules and/or vectors of the invention. Accordingly, in an embodiment, therapeutic RNA molecules and/or vectors according to the present invention are used as a medicament for treating breast cancer, e.g., metastatic breast carcinoma, prostate cancer, e.g., metastatic prostate carcinoma, and/or skin cancer, e.g., metastatic squamous cell carcinoma. In another embodiment, therapeutic RNA molecules and/or vectors according to the present invention are used as a medicament for treating lung cancer, renal cancer, and/or colon cancer.

As reported herein, increased DRR expression activates Akt in tumor cells. Akt activation has been associated with many different cancers including, for example, breast cancer, prostate cancer, skin cancer, melanoma, pancreatic cancer, ovarian cancer, colorectal cancer, lung cancer, colon cancer, and renal cancer (see, e.g.,Cidado and Park, J. Mammary Gland Biol. Neoplasia, 2012; Arker et al., Clin. Cancer Res., 15: 4799-4805,2009; deSouza et al., Curr. Cancer Drug Targets, 9: 163-175, 2009; Davies, Cancer J. 18: 142-147, 2012; Gaikwad and Ray, Am. J. Nucl. Med. Mol. Imaging 2: 418-431 , 2012). Akt, and PI3K/Akt pathways in general, are widely accepted targets for cancer therapeutics. Thus, the finding that increased DRR expression activates Akt in tumor cells suggests a role for therapeutic RNAs of the invention in treating a wide range of cancers for which Akt activation has been implicated in pathogenesis of the disease. Accordingly, it is contemplated that therapeutic RNA molecules and/or vectors according to the present invention may be used as a medicament for treating cancers or tumors associated with Akt activation, such as, for example, metastatic or invasive breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, renal cell carcinoma, or colon carcinoma.

In an embodiment, Akt phosphorylation and/or activation is inhibited by therapeutic RNA molecules and/or vectors and methods of the invention. In another embodiment, tumor or cancer cell invasiveness is inhibited by therapeutic RNA molecules and/or vectors and methods of the invention. In an embodiment, cancer progression is delayed or inhibited. In another embodiment, metastasis is inhibited.

Therapeutic RNA molecules and/or vectors according to the present invention may be used alone or in combination with other cancer therapies. Non-limiting examples of other cancer therapies include resection of the cancer, chemotherapy, radiation therapy, immunotherapy, and/or gene- based therapy. The term "resection" refers to surgical removal or excision of part or all of a tumor. The term "radiation therapy" refers to treatment of cancer using radiation. The term "chemotherapy" refers to treatment of cancer with chemical substances, so-called chemotherapeutics. The term "immunotherapy" as used herein refers to stimulation of reactivity of the immune system towards eliminating cancer cells by using immunotherapeutics. The term "gene-based therapy" refers to treatment of cancer based upon transfer of genetic material (DNA, or possibly RNA) into an individual. Examples of such other cancer therapies include: chemotherapeutics including but not limited to temozolomide, vincristine, vinorelbine, procarbazine, carmustine, lomustine, taxol, taxotere, tamoxifen, retinoic acid, 5-fluorouracil, cyclophosphamide and thalidomide; immunotherapeutics such as but not limited to activated T cells and pulsed dendritic cells; gene transfer of CD3, CD7 and CD45 in glioma cells, concomitantly with delivery of an RNA molecule as defined herein.

Therapeutic RNA molecules and/or vectors according to the present invention may be administered alone or in combination with one or more additional cancer therapy. The latter can be administered before, after or simultaneously with administration of RNA molecules and/or expression vectors.

A further object of the present invention are pharmaceutical preparations which comprise a therapeutically effective amount of an antisense or RNAi molecule and/or expression vector of the invention and a pharmaceutically acceptable carrier. The term "therapeutically effective amount" as used herein means that amount of RNA molecule(s) and/or expression vector(s) that elicits a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

In another embodiment, the invention therefore relates to a pharmaceutical composition for treatment of cancer, preferably glioma, and more preferably glioblastoma, comprising an RNA molecule and/or expression vector according to the invention, and a pharmaceutically acceptable carrier. In yet another embodiment, the invention relates to a pharmaceutical composition for delay of progression of cancer, preferably glioma, and more preferably glioblastoma, comprising an RNA molecule and/or expression vector according to the invention, and a pharmaceutically acceptable carrier. In a further embodiment, the invention relates to a pharmaceutical composition for inhibition of cancer invasion, preferably glioma, and more preferably glioblastoma, comprising an RNA molecule and/or expression vector according to the invention, and a pharmaceutically acceptable carrier. In one embodiment, the invention relates to a pharmaceutical composition for inhibition of malignant glial cell (MGC) invasion.

In a further embodiment, the invention relates to a pharmaceutical composition for treatment of a metastatic or invasive cancer, such as breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, colorectal carcinoma, or renal cell carcinoma. In an embodiment, the invention relates to a pharmaceutical composition for inhibition of Akt phosphorylation or activation in a cancer or tumor cell.

The pharmaceutical composition according to the invention may further comprise at least one additional cancer therapeutic, as discussed above.

The pharmaceutical composition according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories. Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion. Other suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or inhalative administration in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants or wafers.

Preparation of pharmaceutical compositions can be carried out as known in the art. For example, a therapeutic RNA and/or an active compound, together with one or more solid or liquid pharmaceutical carrier substances and/or additives (or auxiliary substances) and, if desired, in combination with other pharmaceutically active compounds having therapeutic or prophylactic action, are brought into a suitable administration form or dosage form which can then be used as a pharmaceutical in human medicine. Pharmaceutical preparations can also contain additives, of which many are known in the art, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.

As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, a carrier is suitable for parenteral administration. Alternatively, a carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. A composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. A carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size in the case of dispersion and by use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in a composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an oligonucleotide of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. A modified oligonucleotide can be prepared with carriers that will protect the modified oligonucleotide against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG) .

Many methods for preparation of such formulations are patented or generally known to those skilled in the art. Sterile injectable solutions can be prepared by incorporating an active compound, such as a therapeutic RNA or an oligonucleotide of the invention, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Therapeutic RNAs and oligonucleotides of the invention may also be formulated with one or more additional compounds that enhance their solubility.

A dosage or amount of a therapeutic RNA and/or expression vector used, alone or in combination with one or more active compounds to be administered, depends on an individual case and is, as is customary, to be adapted to individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, weight and individual responsiveness of the human or animal to be treated, on the efficacy and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the therapeutic RNA and/or expression vector. It shall be understood that dosing and administration regimens are within the purview of the skilled artisan.

In another embodiment, the invention provides a kit comprising a RNA therapeutic or expression vector or a pharmaceutical composition according to the invention, and instructions for use thereof.

Therapeutic Methods

Without intending to be limited by theory, it is contemplated that therapeutic benefits of knocking-down and thus significantly reducing DRR expression in tumor cells may be mediated by inhibiting invasion of a tumor into the brain, for example by inhibiting malignant glial cell (MGC) invasion, so as to reduce or delay cancer invasion into adjacent healthy tissues (e.g., the brain in the case of glioma), based on our novel and unexpected findings that DRR is highly expressed in an invasive component of malignant gliomas and drives MGC invasion in both in vivo and in vitro invasion assays.

It is further contemplated that therapeutic benefits of reducing DRR expression in tumor cells may be mediated by inhibiting invasion of a tumor into other tissues, for example by inhibiting metastasis of breast, prostate, squamous cell, lung, colon or renal cancer, or in any cancer associated with upregulation of DRR expression and/or Akt phosphorylation or activation.

Cell invasion requires a cycle of events that utilizes both the actin cytoskeleton and MTs along with continuous formation and disassembly of FAs. The role of MTs in FA turnover has been well established, and accumulating evidence suggests that MTs grow towards FAs in a process that is coordinated by F-actin. Much less is known about constituents of FAs in normal glial cells and gliomas, and the molecular mechanisms that regulate FA turnover in normal glial cells and gliomas have not been described.

Our findings reported herein suggest that de novo expression of DRR observed in invasive MGCs leads to more rapid FA disassembly and thus invasion. The inability of MGCs to separate from a tumor mass when DRR- actin association is abolished has clinical implications. Indeed, a CD151 - specific metastasis blocking monoclonal antibody has been shown to inhibit metastasis by preventing cell rear retraction and thus cell detachment and migration from the primary tumor mass (Zijlstra et al., Cancer Cell 13:221 - 34.2008).

Malignant glioma invasion is a primary cause of brain cancer treatment failure. We report herein development of a novel functional screening strategy and identification of downregulated in renal cell carcinoma (DRR) as a regulator of invasion. We show herein that: DRR drives invasion in vitro and in vivo; although not expressed in normal glial cells, DRR is highly expressed in an invasive component of gliomas; DRR associates with and organizes actin and microtubular cytoskeletons; and these associations of DRR with actin and microtubular cytoskeletons are essential for focal adhesion (FA) disassembly and cell invasion. Our results provide evidence in support of the view that MTs facilitate FA disassembly and identify DRR as a new player in this normal physiologic process. We have shown that DRR is a novel actin/MT crosslinker that regulates FA disassembly. In support, we show that DRR localizes to the actin cytoskeleton and FAs and interacts with the LC2 subunit MAPI A. We show that DRR expression organizes both the actin and MT cytoskeletons so that MTs approach FAs and promote their disassembly. DRR deficiency, or the disruption of this complex by abolishing DRR-actin or DRR-LC2 association, leads to a loss of coordination between actin and MTs, as well as the inability of MTs to reach FAs. These findings identify DRR as a new cytoskeletal crosslinker that regulates FA dynamics and cell movement. In view of the above, the invention provides a method for treating cancer, such as glioma, for example glioblastoma, in a subject in need thereof, comprising administering a therapeutic RNA of the invention, a vector or a pharmaceutical composition as described herein to said subject. In another embodiment, the invention relates to a method for delaying progression of cancer, such as glioma, for example glioblastoma, in a subject in need thereof, comprising administering a therapeutic RNA, a vector or a composition as provided herein to said subject. The term "subject" as used herein preferably refers to a human, but veterinary applications are also in the scope of the present invention targeting for example domestic livestock, laboratory or pet animals.

The invention further provides methods for down-regulating DRR expression, for example decreasing DRR expression by more than 50%, by more than 70%, or by more than 90%. In an embodiment, DRR expression is decreased or reduced by about 50%, about 60%, about 70%, about 80%, or about 90%. In another embodiment, the invention relates to a method for inhibiting or reducing migration or invasiveness of tumor cells, preferably cells of glioma such as glioblastoma, comprising administering a therapeutic RNA, a vector or a composition of the invention to a subject in need thereof.

The invention further provides methods for inhibiting Akt activation or phosphorylation, for example inhibiting Akt activation by more than 50%, by more than 70%, or by more than 90%. In an embodiment, Akt activation is decreased or reduced by about 50%, about 60%, about 70%, about 80%, or about 90%. In another embodiment, the invention relates to a method for inhibiting or reducing migration or invasiveness of tumor cells, preferably cells of metastatic or invasive breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma, comprising administering a therapeutic RNA, a vector or a composition of the invention to a subject in need thereof.

In other embodiments, the invention provides a method for treating cancer, such as metastatic or invasive breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, and renal cell carcinoma, in a subject in need thereof, comprising administering a therapeutic RNA of the invention, a vector or a pharmaceutical composition as described herein to said subject. In another embodiment, the invention relates to a method for delaying progression of such a cancer in a subject in need thereof, comprising administering a therapeutic RNA, a vector or a composition as provided herein to said subject. In another embodiment, the invention relates to a method for inhibiting or reducing migration or invasiveness of tumor cells for such a cancer, comprising administering a therapeutic RNA, a vector or a composition of the invention to a subject in need thereof.

The invention further provides a method for enhancing efficacy of cancer therapies for treatment of cancer, in particular glioma (preferably glioblastoma), or metastatic or invasive breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma, selected from the group comprising resection, chemotherapy, radiation therapy, immunotherapy, and/or gene therapy, comprising administering a therapeutic RNA molecule, a vector or a composition as defined herein, and simultaneously, separately or sequentially administrating said cancer therapy. The term "enhancing efficacy of a cancer therapy", as used herein, refers to an improvement of conventional cancer treatments and includes reduction of the amount of an anti-cancer composition which is applied during conventional cancer treatment, e.g. amount of radiation in radiotherapy, of chemotherapeutics in chemotherapy, of immunotherapeutics in immunotherapy or of vectors in gene based therapies, and/or to an increase in efficacy of a conventional therapy and an anti-cancer composition when applied at conventional doses or amounts during conventional cancer therapy. In one embodiment, enhancing efficacy of a cancer therapy refers to prolonging survival rate of subjects receiving a therapy.

There is also provided herein the use of DRR as a biomarker for invasive brain cancer cells. Detection of elevated DRR expression can be used to identify invasive tumor cells and for diagnosis and/or prognosis of a tumor, based on DRR expression. Accordingly methods for diagnosis and prognosis of malignant glioma are provided, along with use of DRR as a biomarker for invasiveness. In one embodiment, there is also provided the use of DRR as a biomarker for EGFR-independent cancer invasion, e.g., EGFR-independent brain cancer invasion.

Kits for use in diagnostic and prognostic applications are also provided. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, a container(s) can comprise a probe that is or can be detectably labeled. A probe can be, for example, an antibody specific for a DRR biomarker or an RNA specifically hybridizing to DRR. A kit can also include a container comprising a reporter- means, such as a biotin-binding protein, e.g., avidin or streptavidin, bound to a detectable label, e.g., an enzymatic, florescent, or radioisotope label. A kit can include all or part of the amino acid sequence of a biomarker protein, or a nucleic acid molecule that encodes such amino acid sequences, or a nucleic acid molecule that binds to mRNA of a DRR biomarker, or a nucleic acid molecule that encodes a nucleic acid molecule binding to mRNA of a DRR biomarker.

A kit of the invention will typically comprise a container as described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on a container to indicate that a composition is used for a specific application. Directions and or other information can also be included on an insert which is included with a kit.

In one embodiment, the invention provides a kit comprising at least one agent that binds DRR protein or DRR mRNA; and instructions for use of the at least one agent for determining invasiveness of cancer cells, e.g., brain cancer cells, in a subject. In summary, the present invention, relates to use of an anti-DRR therapeutic approach to treat malignant gliomas as well as other invasive or metastatic cancers. The present therapeutic approach is based on the use of anti-DRR tools relating to RNA interference-(RNAi), antisense-, viral-vector-, or any other related approaches aiming to knock-down DRR expression in human tumor cells. The technical feasibility of the present approach is further illustrated by means of the following non-limiting examples.

EXAMPLES

The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.

Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. MATERIALS AND METHODS

Functional Screening Assay

A normal human adult brain cDNA library (Clontech) was subcloned into the pLib retroviral vector (Clontech) and used to transfect the PT67 packaging cell line using Lipofectamine PLUS reagent (Clontech). The secreted replication deficient retrovirus was collected from the supernatant 24-72 hours post transfection and used to consecutively transduce, over a 72 hour time course, the WT-U251 glial cell line (Fig. 1A).

Human Glioma Analysis

Human glioma samples were obtained from the Brain Tumor Research Centre Tissue Bank at the Montreal Neurological Institute and Hospital (Montreal, Quebec, Canada). Written consent was obtained from all patients and the project was approved by the Institutional Ethics Board at the Montreal Neurological Institute and Hospital.

Cell Extraction from Tissues

Tissues obtained from the operating room (OR) were first washed twice with Phosphate Buffered Saline 1 x (PBS) before being transferred to cell culture Petri dishes where the neurosurgeon separated necrotic tissues and blood vessels from the tumor with a blade. Tissues were then cut, using a blade, into very small pieces and incubated with 5ml of 1 .25% of trysin- EDTA for 30 minutes at 37°C. After that, 7.5 ml of cell culture media (Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 250 U/mL penicillin G, 250 μg/mL streptomycin sulfate, and 4.4 μg/mL amphotericin B (Fungizone)) was added to the sample to neutralize trypsin-EDTA and the sample was transferred to a cell strainer. The rubber of a syringe plunger was used to press the tissue through the cell strainer. 5ml of cell culture media was added to the cell strainer and sample was again pressed through the cell strainer with the rubber of a syringe plunger. This step was repeated twice to maximize cell recovery from the cell strainer. The material was then centrifuged for 20 minutes at 1000 rpm at 4°C to pellet cells. The supernatant was discarded and cells were resuspended with 3ml of erythrocyte lysing buffer (155 mM NH CI / 5.7 mM K₂HP0 / 0.1 mM EDTA, pH = 7.3). The cell mixture was incubated for 15 minutes at RT and then 15 ml of culture media was added to the sample, mixed thoroughly and mix was then centrifuged for 10 minutes at 1000 rpm. The supernatant was discarded and cells were resuspended in culture media. Cells were then allowed to grow for a minimum of 2 weeks in DMEM supplemented with 20% FBS, 2X antibiotics before being used in experiments. After 2 weeks, cells were cultured in DMEM supplemented with 10% FBS and 2X antibiotics. Cultures were maintained at 37°C in a humidified atmosphere of 5% C0₂. These cells are referred to herein as "GBM6" cells. Upon reaching confluency, cells were trypsinized using 0.05% trypsin- EDTA.

Cell Lines U251 human oligodendroglioma cell line (WT), human glial tumor cell line (U343MG), rat astrocytoma cell line (C6), DRR^" and DRR⁺ cells were cultured in DMEM high-glucose supplemented with 10% FBS and a penicillin- streptomycin antibiotic mixture. Human glioblastoma cell line (U87MG) (Cavanee lab, University of California at San Diego) were grown in DMEM high-glucose supplemented with 10% inactivated FBS and a penicillin- streptomycin antibiotic mixture.

DRR^" cell lines were generated using short hairpin RNAs (Paddison et al., 2002) and retroviral transduction. The distal C-terminal sequence

(GCTCTCTCTCTTCGCCGGCCAATGCGGCA) was used to generate the short hairpin loop. RT-PCR was used to confirm reduced DRR mRNA levels and western blotting was used to demonstrate reduced protein levels (Fig. S1 ). DRR^APEPE and DRR^AHRE constructs were generated using the

Strategene QuikChange Site-Directed Mutatgenesis kit. DRR⁺, DRR^APEPE and DRR^AHRE stable cell lines were generated by transfecting WT-U251 cells with DRR, DRR^APEPE or DRR^AHRE expression vectors using Lipofectamine 2000 following the manufacturer's protocol. 72 hours post-transfection cells were expanded and selected in DMEM supplemented with 0.6 mg/ml of G418 for 2 weeks. The resistant colonies were trypsinized and expanded in the selection media. E18-19 rat hippocampal neurons were a generous gift from Dr. P. McPherson (McGill University). Cells were fed every seven days with Neurobasal medium supplemented with B-27, N-2, l-glutamine (500 μΜ) and penicillin/streptomycin (100 units/ml) (Invitrogen).

GBM6 cells were prepared by extraction from tissues as described above.

"MNI 1 " cells are primary brain cancer cells grown directly from a patient's tumor using methods as described above.

Antibodies and Reagents

Affinity-purified rabbit polyclonal anti-DRR antibody directed against amino acids 67-92 was generated by Covance. Mouse anti-vinculin and mouse anti-tubulin antibodies, nocodazole, and G418 were purchased from Sigma. Rat anti-tubulin and mouse anti-GFAP antibodies were purchased from Chemicon. Rhodamine-phalloidin, rabbit anti-FAKpY and Alexa 488-, 694-, and 647-conjugated secondary antibodies, and lipofectamine 2000 were purchased from Invitrogen. Chicken anti-MAP2 antibody was purchased from Encor Biotechnology Inc. GFP-paxillin cDNA plasmid was a generous gift from Dr. I.R. Nabi (University of British Columbia).

Human Glioma Immunolabelling

Fixed paraffin-embedded tissue was sectioned at 5 μιη and mounted. Slides underwent heat-induced epitope-retrieval in citrate buffer (pH 6.0, Lab Vision), at 120°C under high pressure for 10 minutes. All labeling was performed at room temperature on a Lab Vision 360 Autostainer. Antibody binding was amplified using Streptavidin or LV-polymer conjugated to HRP, and visualized using AEC chromogen (Lab Vision). All sections were counterstained with Surgipath 560 hematoxylin, and mounted with Aquatex. DRR antibody controls include antigenic peptide competition,

immunolabelling with preimmune serum and single secondary antibody immunolabelling.

Cell Proliferation, Migration and Invasion Assays

Cells were trypsinized and counted using the Coulter Z Series counter (Beckman-Coulter, Inc.). Measurements were taken twice for two samples of the same cell line and averaged. Cells were plated in a 6-well plate and counted after 24, 48, 72 and 96 hours. To assess 2D cell migration, cells were grown to confluency and a scratch was generated using a pipette tip. Images were captured at regular intervals 1 -1 1 hours post scratch. Tumor spheroids were generated using the hanging drop method and implanted in a collagen type 1 matrix as previously described (Werbowetski-Ogilvie et al., Cancer Research 66: 1464-1472, 2006). The implanted spheroids were imaged after the following time points (0, 24, 48 and 72 hours). Invading areas were measured by calculating the extreme diameter at 4 different angles and by subtracting the extreme diameter of the spheroids at time zero. All experiments were performed in triplicate and are from 3 independent experiments. For the scratch assay shown in Figure 22, the following procedure was used: Following 72 hours post-transfection, cells have reached a monolayer. A 200μΙ pipette was used to perform a scratch. Cells were rinsed 3 times with PBS and fresh media was added to the cells. Images were captured with a 5x objective at the beginning of the scratch and at 24 hours and 48 hours. For each image, distances between one side of the scratch and the other were measured. The distance (μιη) of cell migration was quantified by measuring the distance of the scratch at each time interval and subtracting it from the distance of the scratch at time zero.

Western Blot and Immunoprecipitation

To determine DRR expression levels in WT, DRR^" and DRR⁺ cells, cells were allowed to grow to 80% confluency, washed in cold PBS and lysed with RIPA buffer or 2% hot SDS. Lysates (30 μ9) were separated on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were probed with rabbit anti-DRR and mouse anti-tubulin antibodies followed by the appropriate HRP-conjugated secondary antibodies (Jackson

ImmunoResearch). The ECL plus™ reagent detection kit was used (Pierce). Immunoprecipitation experiments were performed in HEK-293 cells as previously described (Angers-Loustau et al., Molecular Cancer Research 2:595-605, 2004).

Mouse Intracerebral Tumor Implantation

All animal experimentation was approved by the Institution's Animal Care Committee and conformed to the guidelines of the Canadian Council of Animal Care. Six week old CD1 nu/nu athymic mice (Charles River, Canada) were anaesthetized by an intraperitoneal injection containing Ketamine, Xylazine and Acepromazine.

The mouse was secured to a stereotactic frame (Kopf Instruments) and a small incision was made in the scalp at the midline. A burr hole was created 0.5 mm anterior and 2 mm lateral to bregma. A microliter syringe (Hamilton Company) was slowly lowered through the burr hole to a depth of 4.4 mm and a cell suspension containing 2x10⁵ cells in 3 μΙ of PBS was injected over 12 minutes. Animals were euthanized at one month post- injection to assess tumour growth and invasion.

Yeast two-hybrid Screening

Yeast two-hybrid screens were performed using the Matchmaker™ Two-Hybrid System 3 (Clontech). Full-length DRR was used as the bait to screen a human brain cDNA library (Clontech).

Immunocytochemistry

Cells were grown on glass coverslips or on fibronectin (1 C^g/ml) coated coverslips, fixed with 4% PFA and permeabilized with 0.5% TritonX- 100 before being immunolabeled. The FA disassembly assay was performed as previously described (Ezratty EJ et al., Nat. Cell Biol. 7:581-590, 2005). Briefly, cells were incubated in serum-free media for 24h before being treated with nocodazole (10μΜ; 4h). The drug was washed out along a variable time course (5, 15, 30 and 60 min) using serum-free media. Fluorescently labeled cells were visualized with a Zeiss 510 confocal microscope (63x objective). The number and surface area of vinculin-stained focal adhesions were quantified using Image J software. At least 10 fields from three independent experiments were quantified.

For immunofluorescence studies, cells were fixed with 4% PFA and permeabilized with 0.5% TritonX-100 before being processed for immunostaining, as described above. Cells were labelled with mouse anti- vinculin to visualize focal adhesions and rhodamine-phalloidin was used to stain actin. Fluorescently labelled cells were visualized with a Zeiss 510 confocal microscope using 63x objectives.

Confocal Videomicroscopy

WT or DRR⁺ cells were seeded on 35 mm glass bottom culture dishes (MatTek Corporation) before being transfected with GFP-paxillin. 24 - 48h post-transfection the images were captured every 1 min for 170 minutes using a Zeiss 510 confocal microscope (63x objective). Five DRR⁺ and five control (WT) cells were analyzed, and a total of 17 FAs were analyzed for DRR⁺ cells. The apparent rate constants for the incorporation of GFP-paxillin into FAs and its disassembly from FAs was quantified using the technique described in Webb et al., 2004. Measurements were obtained from five cells, 5-10 FAs/cell. In control cells, no FAs were identified that assembled or disassembled within the 170 minute imaging interval. Data is presented as mean ± standard error.

Primary Culture of Human Glioma

Tissues obtained from surgical resection were rinsed two times with Phosphate Buffered Saline 1x (PBS) before being transferred to cell culture dishes. Necrotic tissues and blood vessels were separated from the tumor. Tissues were then cut and incubated with 5ml of 1 .25% of trysin-EDTA for 30 minutes at 37°C, after which 7.5 ml of cell culture media (Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 250 U/mL penicillin G, 250 μg/mL streptomycin sulfate, and 4.4 μg/mL amphotericin B (Fungizone)) was added to the sample to neutralize trypsin- EDTA. The sample was then transferred through to a cell strainer. 5ml of cell culture media was added to the cell strainer and the process was repeated three times. The suspension was then centrifuged for 20 minutes at 1000 rpm at 4°C to pellet the cells. The supernatant was discarded and the cell pellet was resuspended with 3ml of erythrocyte lysing buffer (155 mM NH CI / 5.7 mM K₂HP0 / 0.1 mM EDTA, pH = 7.3). The cells were incubated for 15 minutes at room temperature and then 15 ml of culture media was added to the sample, mixed thoroughly and centrifuged for 10 minutes at 1000 rpm. The supernatant was discarded and cells were resuspended in culture media. After which, cells were allowed to grow for a minimum of 2 weeks in DMEM supplemented with 20% FBS, 2X antibiotics before use. After 2 weeks, cells were cultured in DMEM supplemented with 10% FBS and 2X antibiotics. Cultures were maintained at 37°C in a humidified atmosphere of 5% C0₂. Upon reaching confluency, cells were trypsinized using 0.05% trypsin- EDTA.

Cell transfection and 3D invasion assay

50 000 cells were plated in a 6 well dish overnight and the next day, they were transfected with plasmid vector expressing GFP alone or DRR antisense using GeneJuice™ (Novagen) according to the manufacturer's instructions (2.5 μg DNA; 4 μΙ GeneJuice ' ^M in 100 μΙ of OptiMEM). 72h post- transfection, cells were detached by adding 0.5 ml of 0.05% Trypsin-EDTA to the wells and incubated for 30 second at 37°C. Cells were then neutralised with 1 ml of culture media, transferred to 15 ml tube and counted.

To generate tumor spheroids, drops (20 μΙ) of cell culture media containing 25 000 cells were suspended from an inverted Petri dish lid. 5 ml of PBS was added at the bottom of the dish to prevent evaporation of the drops. To form cell aggregates, the hanging drops were incubated for 72h at 37°C. The aggregates were transferred to 2% agar/PBS (pH 7.4) coated Petri dishes containing 10 ml culture media and were incubated at 37°C for another 48h period to allow the aggregates to become round like a spheroid. Then, spheroids were implanted into a liquid collagen Type I solution (2.5 mg/ml 0.012N HCL) mixed with 10x DMEM and 0.1 mM NaOH at a ratio of (8: 1 :1 ). Collagen-containing spheroids were allowed to solidify at 37°C for 30 min after which 0.5 ml of tissue culture media was added to each well. The implanted spheroids were imaged after the following time points: 0, 24, 48, 72 and 96 hours. Invading areas were measured by calculating the extreme diameter at 4 different angles and by subtracting the extreme diameter of the spheroids at time zero.

For experiments in Figures 17-21 , transfection was carried out as follows: The day before transfection, cells were plated so as to reach 75% confluency at the time of transfection. Cell media was replaced with fresh media before transfection. Complexes of DRR oligomer and lipofectamine 2000 (Invitrogen) were prepared according to the manufacturer's instructions. Briefly, lipofectamine was gently mixed in opti-MEM and left at room temperature for 5 min. DRR oligomer was first mixed with opti-MEM (so that the final concentration added to the cells was 20nM) and gently mixed with lipofectamine. Lipofectamine-DRR oligomer complexes were incubated at room temperature for 20 minutes before being added to the cells. The day after transfection, fresh cell media was added to the transfected cells. Cells were fixed or lysed following 72 hours post-transfection.

Antisense oligonucleotide synthesis Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides (Damha and Ogilvie, 1993, In Agrawal, S. (ed.), Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology, Vol. 20, The Humana Press Inc., Totowa, NJ, pp/ 81 -1 14). Syntheses were performed on an Applied Biosystems 3400 DNA Synthesizer at a 1 μιηοΙ scale using Unylink CPG support (ChemGenes). All phosphoramidites were prepared as 0.15M solutions in acetonitrile (ACN), except DNA, which was prepared as 0.1 M. 5-ethylthiotetrazole (0.25M in ACN) was used to activate phosphoramidites for coupling. Detritylations were accomplished with 3% trichloroacetic acid in CH₂CI₂ for 1 10s. Capping of failure sequences was achieved with acetic anhydride in tetrahydrofuran (THF) and 16% N- methylimidazole in THF. Sulphurizations were accomplished using a 0.1 M solution of xanthane hydride in 1 :1 v/v pyridine/ACN. Coupling times were 1 10s for DNA amidites (270s for guanosine), and 600s for 2'F-ANA phosphoramidites, with the exception of guanosine phosphoramidites which were allowed to couple for 900s. Deprotection was accomplished with an on- column decyanoethylation step using anhydrous 2:3 TEA:ACN in three 15min washes followed by an ACN wash. Deprotection and cleavage from the solid support was accomplished with either 3: 1 NH40H:EtOH for 48h at room temperature (RT), or with 40% methylamine for 10 min at 65°C (Bellon, L, 2000, Curr. Protocols. Nucleic Acid Chem., 3.6.1 -3.6.13).

Purification of crude oligonucleotides was done either by preparative denaturing polyacrylamide gel electrophoresis (PAGE) using 24% acrylamide gels, or by reverse phase HPLC on a Waters 1525 HPLC using a Varian Pursuit 5 reverse phase C18 column with a stationary phase of l OOmmol triethylammonium acetate in water with 5% ACN, and a mobile phase of HPLC-grade acetonitrile. Gel bands were extracted overnight in DEPC- treated autoclaved Millipore water, and lyophilized to dryness. All purified oligonucleotides were desalted with Nap-25 Sephadex columns from GE Healthcare. Sequences were verified by ESI-LCMS. Sequences targeting DRR mRNA were designed using the published mRNA sequences available on the NCBI website. Effective antisense sequences targeting DRR were designed using an antisense oligonucleotide (AON) sequence selection tool available from IDT

(http://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx7sour ce=menu). Predicted antisense sequences were checked using BLAST alignment tools (NCBI) to check for absence of off-target hits. Additionally, mRNA secondary structure of a section of the DRR mRNA was predicted using the MFOLD tool (http://mfold.rna. albany.edu/?q=mfold/RNA-Folding- Form) to look for mRNA-accessibility for AON binding. AONs targeting DRR are shown in Table 1 (G4 (SEQ ID NO: 14), G5 (SEQ ID NO: 15), and G6 (SEQ ID NO: 16)), and control AONs not targeting DRR were also prepared for control experiments (G1 (SEQ ID NO: 1 1 ), G2 (SEQ ID NO: 12), G3 (SEQ ID NO: 13)). In addition to the DRR-targeting AON sequences provided herein, e.g., in Table 1 , other DRR-targeting AON sequences could be selected using these methods by choosing sequences complementary to other DRR mRNA regions, while ensuring specificity for DRR mRNA but not other mRNA sequences. It should be understood that any antisense molecule, e.g., antisense oligonucleotide, which targets DRR and reduces or decreases DRR expression is encompassed.

siRNA oligonucleotide synthesis

Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides (Damha, M.J. and Ogilvie.K.K. (1993), In Agrawal.S. (ed.), Protocols for

Oligonucleotides and Analogs: Synthesis and Properties, Methods in

Molecular Biology, Vol. 20, The Humana Press Inc., Totowa, NJ, pp. 81- 1 14).

Syntheses were performed on an Applied Biosystems Synthesizer on controlled pore glass at a 1 μιηοΙ scale. All phosphoramidites were prepared as 0.15M solutions in dry acetonitrile (ACN), except DNA, which was prepared as 0.1 M. 5-ethylthiotetrazole (0.25M in ACN) was used to activate the phosphoramidites for coupling. Detritylations were accomplished with 3% trichloroacetic acid in CH₂CI₂ (DCM) for 1 10s. Capping of failure sequences was achieved with acetic anhydride in tetrahydrofuran (THF) and 16% N- methylimidazole in THF. Oxidation was done using 0.1 M l₂ in 1 :2: 10 pyridine:water:THF. Coupling times were 600s for RNA, 2'F-ANA, and 2'F- RNA phosphoramidites, with the exception of their guanosine

phosphoramidites, which were allowed to couple for 900s. Cyanine 5 phosphoramidites (Glen Research) were coupled at 0.1 M concentration using a manual coupling step carried out under anhydrous conditions on- column between two 1 ml_ syringes containing activation reagent and Cyanine 5 phosphoramidite in acetonitrile (CAN) (capping, oxidation, and dethtylations were done as usual on the DNA synthesizer, with the exception that a 0.02M oxidation solution was used in place of the regular 0.1 M solution). 5'- phosphorylation of chemically modified antisense strands was achieved using bis-cyanoethyl-N, N-diisopropyl-2-cyanoethyl phosphoramidite at 0.15 M (600s coupling time). Deprotection and cleavage from the solid support was accomplished with either 3: 1 NH OH:EtOH for 48h at room temperature (RT), or with 40% aqueous methylamine for 10 min at 65°C.

Oligonucleotides containing RNA were synthesized with standard 2'- TBDMS phosphoramidites, and desilylation was achieved with either neat triethylamine trihydrofluoride for 48h at ambient temperature, or with triethylamine trihydrofluoride/N-methyl pyrrolidinone/triethylamine (1 .5:0.75: 1 by volume) for 2.5h at 65°C. Purification of crude oligonucleotides was done either by preparative denaturing polyacrylamide gel electrophoresis (PAGE) using 24% acrylamide gels, or by reverse phase HPLC on a Waters 1525 HPLC using a Varian Pursuit 5 reverse phase C18 column with a stationary phase of l OOmmol triethylammonium acetate in water with 5% ACN (pH 7), and a mobile phase of HPLC-grade acetonitrile (Sigma). Gel bands were extracted overnight in DEPC-treated autoclaved Millipore water, and lyophilized to dryness. All purified oligonucleotides were desalted with Nap- 25 Sephadex columns from GE Healthcare.

siRNAs were prepared by annealing equimolar quantities of complementary oligonucleotides in siRNA buffer (100 mM KOAc, 30 mM HEPES-KOH, 2 mM Mg(OAc)₂, pH 7.4) by slowly cooling from 96°C to RT in a ceramic heat block. Sequences targeting DRR mRNA (isoforms 1 and 2) were designed using the published mRNA sequences available on the NCBI website. mRNAs were submitted to the Whitehead siRNA design tool (http://jura.wi.mit.edu/bioc/siRNAext/), and siRNA sequences targeting the open reading frame of the mRNA were selected. The selected sequences have less than 15 % identity with other cellular mRNAs. From the sequence selection tool output, the sequence shown below in Table 1 was chosen for chemical modification with successful chimeric designs identified previously (Deleavey, G.F., et al. (2010) Nucleic Acids Research, 38, 4547-4557).

siRNAs siRNAI , siRNA2, and siRNA3 target DRR mRNA, whereas Control is a scrambled control.

RESULTS

Functional Screening Assay Identifies DRR as a Promoter of Invasion

MGC invasiveness can be assayed using a 3D invasion model (Del Duca, D. et al., Journal of Neurooncology 67:295-303, 2004). Using this model as a starting point, we developed a novel functional screening assay by retrovirally transducing MGCs, the U251 glioma cell line, to express an entire brain cDNA library. We reasoned that if we could make a cell heterologously express a gene that promotes invasion, it would be distinguishable from other cells as a hyperinvasive cell. Tumor spheroids were generated from the transduced MGCs and their invasiveness was assessed in the 3D invasion model. Distinguishable hyperinvasive cells were then captured and expanded in culture and the originally transduced gene was identified (Fig. 1 A). DRR was identified as a strong promoter of invasion using this forward genetic approach.

To test whether or not DRR acts as an effector of MGC invasion, we generated composite tumor spheroids made up of both DRR-overexpressing MGCs (DRR⁺, Fig. 8) and wild-type (WT) MGCs, and studied the invasion parameters of each cell line (Fig. 1 B-E). While MGCs endogenously express DRR, DRR⁺ cells invade 240% farther than WT cells. By contrast, reducing DRR expression in MGCs using RNA interference (DRR^", Fig. 8) causes a significant decrease in invasion (Fig. 1 F, G & J). To test if reducing DRR expression decreases invasion in other glial cell lines, we developed U343- DRR^", C6-DRR^" and U87MG-DRR^" and stable cell lines and tested their invasiveness. Compared to their wild-type counterparts which express endogenous DRR, all DRR^" cell lines exhibited a significant reduction in their invasiveness (Fig. 1 1 C-E). Interestingly, DRR expression also leads to a profound change in cell morphology as DRR⁺ cells are elongated and spindle shaped whereas DRR^" cells are round (Fig. 1 H, I, & K). Experiments in 2D migration assays also reveal differences in the morphology of cells as they migrate. DRR⁺ cells migrate with long thin protrusions whereas WT and DRR^" cells migrate with a uniform broad lamella (Fig. 9). An elongated spindle cell shape has been shown to be the preferred mode of MGC movement through brain (Beadle et al., Mol Biol Cell. 19:3357-68, 2008).

We next examined DRR's role as an invasion promoter in a mouse model. DRR⁺ and DRR^" tumors were implanted into the subcallosal/caudate region of mice and invasion was assessed (Fig. 1 L & M). DRR^" tumors grow as a well circumscribed mass without invasion into the adjacent parenchyma, and these cells have a round morphology. Conversely, DRR⁺ tumors are highly invasive. These invasive cells, which are distinguished by their large, hyperchromatic and elongated nuclei, have an elongated shape, separate from the tumor mass, invade parenchyma, and, importantly, move towards and into the corpus callosum. Invasion into white matter tracts such as the corpus callosum is a preferred invasion paradigm used by human malignant glial tumors (Pedersen et al., Int. J. Cancer, 62:767-71 , 1995). Furthermore, DRR⁺ tumors were smaller than DRR^" tumors (Fig. 8B, C) suggesting a decrease in cell proliferation as previously described (Wang et al., Genes Chromosomes Cancer 27: 1 -10, 2000). We assessed the role of DRR in cell division and also found that cell division is inversely correlated with DRR expression (Fig. 1 N). The notion that MGC invasion and proliferation are temporally exclusive events has been described (Giese et al. , Int J Cancer 67:275-282.1996).

Thus, we have identified DRR as a regulator of cell movement and a driver of cell invasion. We have validated this finding in both in vitro and in vivo invasion assays, confirming the importance of DRR for cell invasion. DRR is Expressed in Neurons and Human Gliomas but not in Normal Glia

To validate clinically our in vitro and in vivo findings, we determined the expression pattern of DRR in normal human brain and malignant gliomas. We found that in normal human brain, DRR is strongly expressed in neurons but not in astrocytes or in oligodendrocytes (Fig. 2A-F, see Fig. 10 for high magnification images). Co-labeling of cultured embryonic rat neurons and glia with neuronal and glial markers also shows that DRR is expressed in neurons but not in glial cells (Fig. 2G-L). DRR antibody controls including antigenic peptide competition, immunolabelling with pre-immune serum and single secondary antibody immunolabelling were negative. Analysis of DRR expression in 8 malignant gliomas of each grade indicates that DRR is highly but not uniformly expressed in all malignant glial tumors (Fig. 2M). Grade 2 and 3 gliomas, which are highly invasive tumors with low proliferation rates, uniformly express DRR. In contrast, grade 4 gliomas, which are both highly invasive and highly proliferative, express DRR in a suggestive pattern. The invasive peripheral tumor cells uniformly express DRR whereas the central proliferative tumor region showed variability in DRR expression. The central tumor in 5 out of 8 grade 4 gliomas showed little to no DRR expression, whereas the central tumor was DRR positive in 3 out of 8 tumors. Taken together, these data show that while DRR is not expressed in normal glial cells, it has a robust and differential expression pattern in malignant gliomas. DRR Associates with the Cytoskeleton

To uncover how DRR functions to drive cell movement, we localized DRR at the sub-cellular level. Endogenous or heterologously expressed DRR predominantly localizes along actin stress fibers, FAs and membrane ruffles (Fig. 3A). In agreement with a previous report (Wang et al., Genes Chromosomes Cancer 27: 1 -10, 2000), DRR can also be found in the nucleus (Fig. 12). These results suggest that DRR may be promoting invasion through a direct influence on the cytoskeletal apparatus or though a regulatory role in the nucleus.

To address this question, we uncoupled DRR from the actin cytoskeleton by identifying minimal domains required for actin association. Sequential N- and C-terminal truncated constructs of human DRR were generated and assayed for localization using fluorescent tags. Two minimal regions capable of actin association were identified, amino acids 62-100 and 108-120 (Fig. 14). We determined that domains required for actin association are conserved across species. We identified and mutated the amino acids within the 62-100 and 108-120 regions that were conserved across human, mouse, rat and zebrafish DRR (Fig. 13). The combined mutation of the conserved proline-glutamate (PE) motifs to alanines in both segments

Apcpc

(DRR ) leads to a significant perturbation of the actin cytoskeleton and abolishes actin association with the remaining stress fibres (Fig. 3A). These findings indicate that cytoskeletal association of DRR is conserved across species.

In order to identify molecules that play a role in DRR association with the cytoskeleton, we used yeast two-hybrid screening of normal brain libraries to identify DRR binding partners. Using this assay, the light chain (LC2) subunit of MAPI A was identified as a candidate DRR binding protein. We have also shown that DRR and LC2 colocalize along actin stress fibers and membrane ruffles, and can be co-immunoprecipitated when

heterologously expressed (Fig. 3B and C), consistent with the association of these proteins. Interestingly, mutation of the DRR actin binding sites appears to increase DRR association with LC2 (Fig. 3B). We also found, in some PEPE

cells, that the non-actin binding form of DRR (DRR ) can localize to microtubules (data not shown).

We then developed non-LC2 binding forms of DRR using truncation and amino acid mutagenesis analysis. A minimal N-terminal histidine- arginine-glutamate (HRE) sequence was found to be required for LC2 binding (Fig. 3B). When this region is mutated, DRR there is a significant perturbation of the actin cytoskeleton and the localization pattern of DRR changes. There is an increase in DRR expression in the nucleus and diffuse cytoplasmic localization (Fig. 3A). Slight actin association was also observed.

In summary, the data show that DRR localizes to the actin

cytoskeleton, FAs and nucleus. Minimal regions required for actin association PFPF

(DRR ) have been defined. The LC2 subunit of MAPI A has been identified as a DRR associated protein, and this association can be disrupted

ΔΗ RE

by mutation of the amino terminal HRE region (DRR ).

DRR association with the Cytoskeleton is Required for Cell Movement

The ability to disrupt the DRR-actin and DRR-LC2 associations provided two approaches to determine if DRR association with the cytoskeleton is required to drive cell movement. Stable cell lines expressing DRR^APEPE or DRR^AHRE were generated and tested for invasiveness in a 3D invasion assay. We found that loss of the DRR-actin association leads to a ~3-fold reduction in invasion compared to WT cells, suggesting that this mutant form of DRR is acting as a functional dominant negative (Fig. 4). A similar finding is also seen when the DRR-LC2 interaction is disrupted (Fig. 4). Together, these data show that DRR association with actin and LC2 is required to drive cell invasion.

DRR Regulates Focal Adhesion Dynamics

The process of cell movement requires regulated FA dynamics (Lauffenburger et al., Cell 84:359-369, 1996; Friedl et al., Nat Rev Cancer 3:362-74, 2003). To determine if DRR expression affects FAs, we expressed a GFP-paxillin fusion protein and studied the effect of DRR expression on FA dynamics using confocal videomicroscopy (Fig. 5). In non-polarized DRR⁺ cells we found that the total time taken for FAs to form and disassemble is 40.05 ± 3.00 minutes (Fig 5 A-C). The rate constant for GFP-paxillin incorporation into FAs was (6.2 ± 0.9) x 10^~3 min^"1 and the rate constant for GFP-paxillin disassembly was (8.6 ± 0.7) x 10^"3 min^"1. Conversely, FAs were not dynamic in WT control cells. We were unable to detect FAs that formed or disassembled within the 170 minute imaging interval (Fig 5D). These data strongly support a mechanism whereby DRR drives cell invasion by enhancing FA dynamics.

It has been established that FA disassembly requires polymerized microtubules (MTs) (Kaverina et al., J. Cell Biol. 142: 181-190,1998; Kaverina et al., J. Cell Biol. 146: 1033-1044, 1999; Krylyshkina et al., J. Cell Biol.

156:349-359, 2002; Krylyshkina et al., J. Cell Biol. 161 :853-859, 2003; Ezratty et al., Nat. Cell Biol. 7:581-590, 2005). Since we have shown that DRR interacts with the LC2 subunit of MAPI A and that DRR overexpression leads to less stable FAs, we examined directly if DRR promotes FA disassembly.

MT control of FA disassembly can be examined using the microtubular depolymerizing agent nocodazole to disassemble microtubules. After nocodazole application, FAs increase in size since there are no microtubules available for disassembly. Upon nocodazole washout, microtubules polymerize and focal adhesions disassemble (Ezratty et al., Nat. Cell Biol. 7:581-590, 2005). We performed this experiment using DRR⁺ and DRR^" cells plated on the extracellular matrix component fibronectin. Two striking observations were made. First, upon nocodazole application and MT depolymerization, DRR⁺ cells develop more and larger focal adhesions compared to non-treated cells (Fig. 6A). In contrast, we did not observe differences in FA number and size in nocodazole treated versus non-treated DRR^" cells (Fig. 6A) or WT cells (data not shown), suggesting that DRR deficiency leads to large and mature FAs. Second, when MTs repolymerize in cells overexpressing DRR, FAs begin to disassemble within 5 minutes whereas FAs in DRR^" cells (or WT cells, data not shown) only begin to disassemble after 15 minutes and do not completely disassemble (Fig. 6A). When the same experiment was performed with the non-actin binding form of DRR (DRR^APEPE) or the non-LC2 form of DRR (DRR^AHRE) the FA

disassembly kinetics were similar to DRR deficiency conditions (Fig. 6B, data not shown for DRR ). The finding that DRR expression promotes FA disassembly whereas DRR deficiency leads to stable mature FAs points to DRR as a novel regulator of FA dynamics.

DRR Organizes the Actin and Microtubular Cytoskeletons

Results from the FA disassembly assay suggest that DRR association with both the actin cytoskeleton and the LC2 subunit of MAPI A are required for FA disassembly (Fig. 6). One mechanism through which DRR could achieve this result is to alter MT dynamics by placing MTs in the vicinity of FAs (Kaverina et al., J. Cell Biol. 146: 1033-1044, 1999). We tested this hypothesis and found that DRR regulates both the MT and actin

cytoskeletons. DRR expression leads to a highly organized MT system that strongly parallels the localization pattern of the actin cytoskeleton (Fig. 7A). In contrast, DRR deficiency leads to an irregular, poorly organized MT cytoskeleton that does not parallel the actin cytoskeleton (Fig. 7B). DRR deficiency also leads to a profound change in the actin cytoskeleton with loss of stress fiber formation and the promotion of a cortical actin system (Fig. 7 A and B). The promotion of a stress fiber actin system allows for actomyosin contraction and thus cell rear retraction (Verkhovsky et al., J. Cell Biol.

131 :989-1002, 1995). Importantly, we also found that DRR expression is required for MTs to reach FAs. MTs in DRR deficient cells do not approach FAs (Fig. 7B). In contrast, DRR expression leads to a close association between MTs and FAs (Fig. 7A). Together, these data strongly suggest that DRR is a novel regulator of FA dynamics by controlling both the actin and MT cytoskeletons (Fig. 7C).

Reduction of DRR expression inhibits human glioma invasion

Human high grade gliomas were surgically resected and immediately placed in culture. Two weeks later they were transfected with a control GFP vector or DRR-RNAi (SEQ ID NO: 1 ) (vector also contains green fluorescent protein (GFP)). Tumor spheroids were generated from these cells and implanted into a collagen matrix. Brightfield (upper lanes) and fluorescence images (lower lanes) were captured at 1 to 14 days post-implantation (Fig. 16). Non-transfected tumors (Fig. 16A) and control GFP-transfected tumors (Fig. 16B) readily invade, whereas DRR-RNAi transfected tumors (Fig. 16C) do not. Fig. 16D shows quantification of invasion distance from spheroid edge.

These results indicate that reduction of DRR expression inhibits human glioma invasion, and represent the first demonstration of DRR as a therapeutic target to inhibit human brain cancer invasion in subjects with primary brain cancers.

Several types of DRR antisense oligonucleotides reduce DRR expression

The efficacy of different DRR antisense oligonucleotides in reducing

DRR expression was compared.

DRR+ cells were transfected with the indicated DRR antisense (Antisense G4 (SEQ ID NO: 14; an altimer), Antisense G5 (SEQ ID NO: 15; a gapmer) or Antisense G6 (SEQ ID NO: 16; a gapmer); a non-targeting control antisense (Ctl Antisense); or left untransfected (Untransfected).

Oligonucleotide G1 (SEQ ID NO: 1 1 ) is a non-targeting altimer control, and oligonucleotides G2 (SEQ ID NO: 12) and G3 (SEQ ID NO: 13) are non- targeting gapmer controls.

DRR expression level was determined 72 hours post-transfection using Western blotting (Fig. 17). The results show that different antisense oligonucleotides are effective at reducing DRR expression.

We next looked at changes in DRR actin's cytoskeletal and focal adhesion following transfection with the different DRR antisense

oligonucleotides (Fig. 18). The results show that reduction of DRR expression by treatment with DRR antisense oligonucleotides induced cells to shift from an elongated spindle morphology to a round morphology. We also found that treatment with DRR antisense oligonucleotides leads to large focal adhesions.

The effect of the different DRR antisense oligonucleotides on DRR+ cell migration was analyzed using an in vitro scratch assay (Fig. 19). The results show that at time zero all conditions are similar, however within 48 hours, the scratch in control DRR+ cells is no longer evident. In contrast, treatment with DRR antisense oligonucleotides prevents the gap from closing. These results demonstrate that reducing DRR expression prevents cell migration.

DRR+ cell invasion was also analyzed using an in vitro 3D invasion assay (Fig. 20). It can be seen that control DRR+ tumor spheroids are highly invasive whereas treatment with DRR antisense oligonucleotides impairs tumor spheroid invasion. Quantification of invasion reveals that treatment with DRR antisense oligonucleotides leads to a significant reduction in invasion (Fig 20B).

Next, we visualized changes in the actin cytoskeletal and focal adhesions of human glioblastoma cells following transfection with the different DRR antisense oligonucleotides (Fig. 21 ). We found that reduction of DRR expression by treatment with DRR antisense oligonucleotides induced cells to shift from an elongated spindle morphology to a round morphology. We also found that treatment with DRR antisense

oligonucleotides leads to large focal adhesions.

Human glioblastoma cell migration was analyzed using an in vitro scratch assay (Fig. 22). The results show that within 24 hours, the scratch in control glioblastoma cells has nearly closed whereas treatment with DRR antisense oligonucleotides prevents the gap from closing. These results demonstrate that reduction of DRR expression prevents glioblastoma migration.

Several types of DRR siRNA oligonucleotides reduce DRR expression

The efficacy of several DRR siRNA oligonucleotides in reducing DRR expression was determined. Altimer oligonucleotides (oligonucleotides with alternating units, such as those described, for example, in PCT publication no. WO/2003/064441 ) and gapmer oligonucleotides (such as chimeric antisense oligonucleotides; such oligonucleotides are described, for example, in PCT publication no. WO/2002/20773) were tested, along with

corresponding non-DRR targeting controls.

DRR+ cells were transfected with a DRR siRNA as indicated (siRNAI (SEQ ID NO: 17/18), siRNA2 (SEQ ID NO: 19/20); or siRNA3 (SEQ ID NO: 23/24; a FANA FRNA altimer)); or a non-targeting control sequence (Ctl siRNA; SEQ ID NO: 25/26).

DRR expression level was determined 72 hours post-transfection using Western blotting in DRR+ cells (Fig. 23). The results showed that siRNA oligonucleotides were effective at reducing DRR expression.

DRR expression level in MNI 1 stem cells was determined 72 hours post-transfection using Western blotting in DRR+ cells (Fig. 24). The results showed that various siRNA oligonucleotides were effective at reducing DRR expression.

We next looked at changes in actin cytoskeletal and focal adhesions in

DRR overexpressing cells and GBM6 cells following transfection with DRR siRNA oligonucleotides (Figs. 25, 26 and 28). The results showed that reduction of DRR expression by treatment with DRR siRNA oligonucleotides induced cells to shift from an elongated spindle morphology to a round morphology. We also found that treatment with DRR siRNA oligonucleotides leads to large focal adhesions (Figs. 25, 26 and 28). Thus, our results indicated that primary glioblastoma cells (GBM6 cells) treated with siRNAI showed changes consistent with a lack of DRR expression including increased cell size, increased focal adhesion size and conversion from a spindle shape morphology to a round cell morphology. The actin cytoskeleton also converted from a stress fibre network to a cortical network.

The effect of the various DRR siRNA oligonucleotides on DRR+ cell migration was analyzed using an in vitro scratch assay (Fig. 27). The results showed that at time zero all conditions were similar, however within 48 hours, the scratch in control DRR+ cells was no longer evident. In contrast, treatment with DRR siRNA oligonucleotides prevented the gap from closing. These results demonstrated that reducing DRR expression prevented cell migration. In summary, we demonstrate herein a novel functional screening assay to identify promoters of invasion, and, using this assay, we have identified DRR as a promoter of invasion. We show here that DRR promotes MGC invasion in 3D cultures in vitro and in mouse models of invasion.

Characterization of DRR expression in normal human brain and gliomas reveals that in normal brain DRR is abundantly expressed in neurons but not in glia. In contrast, DRR is uniformly and highly expressed in the invasive regions of both low and high grade gliomas, whereas its expression in the central proliferative region of high grade gliomas is variable. We also demonstrate that reduction of DRR expression inhibits human glioma invasion.

Together, these findings indicate that DRR is an important regulator of glioma invasion and a target for therapeutic treatment of glioma. In addition, DRR is a useful biomarker to delineate invasive regions and grade malignant gliomas.

We note that recent studies by others have linked DRR and malignant gliomas, reporting that DRR expression is reduced in high grade gliomas compared to low grade gliomas, while expression in normal brain was not described (van den Boom et al., Int. J. Cancer 1 19:2330-2338, 2006). An anti-proliferative function of DRR was also reported elsewhere (Wang et al., Genes Chromosomes & Cancer 27: 1 -10, 2000). In contrast to these reports of a role for DRR as a tumour suppressor, we report herein the surprising and unexpected finding that DRR acts as a driver of invasion in tumor cells and is highly expressed in the invasive regions of malignant gliomas. We show for the first time that DRR expression is associated with an invasive phenotype in glioma cells and that reduction of DRR expression inhibits human glioma invasion. Thus our results suggest for the first time a central role for DRR in glioma biology, as a driver of cell invasion as well as a regulator of cell proliferation.

DRR is involved in the EFGR/PI3K-PTEN/Akt pathway

Since the EFGR/PI3K-PTEN/Akt pathway has been shown to be a driver of GBM invasion that is altered in over 80% of GBMs and pAkt is elevated in a high percentage of GBMs, and, as reported herein, DRR is overexpressed in invasive gliomas compared to normal glial cells, we tested whether DRR is involved in the EFGR/PI3K-PTEN/Akt pathway. As reported below, we found that DRR expression leads to elevated Akt activation by recruiting Akt to FAs in an adhesion and src family kinase (SFK) dependent manner. This augmented Akt activation leads to NFkB activation and transcription of MMPs involved in glioma invasion. We also found that reduction of DRR expression using antisense oligonucleotides prevented glioma invasion in a xenograft mouse model. Thus, DRR represents a novel GBM target and therapeutic RNA molecules provided herein, e.g., DRR antisense oligonucleotides, are a novel therapeutic approach to prevent brain cancer invasion.

The Materials and Methods for the below experiments relating to the

EFGR/PI3K-PTEN/Akt pathway (data shown in Figures 29-34) are as follows:

Antibodies and reagents: Anti-phospho-AKT (Ser473), anti- phospho-AKT (Thr308), anti-AKT, anti-phospho-p44/42 MAPK

(Thr202/Tyr204), anti-p44/42 MAPK, anti-SRC, anti-ILK1 and Signal Silence ILK1 siRNA II (Cell Signaling Technology, Danvers, MA) and anti-EGFR (Santa-Cruz Biotechnology Inc., Santa Cruz, CA) were used. Anti- phosphotyrosine, anti-FAK, anti-phospho-SRC (Tyr418) (Millipore, Billerica, MA) were used. Anti-phospho-FAK (Tyr397), anti-phospho-SRC (Tyr418) (Invitrogen, Burlington, ON) were used. Anti-DRR (Covance, Princeton, NJ), anti-a-tubulin, anti-a-vinculin and fibronectin from bovine plasma (Sigma- Aldrich, St. Louis, MO) were used. AG1478, U0126, LY294002, wortmannin, PP2, PF-228, GRGDSP peptide (Calbiochem, Merck KGaA, Darmstadt, Germany), C3 transferase (Cytoskeleton Inc, Denver, CO) were used. Texas Red EGF (Invitrogen) was used. PureCol® Bovine Collagen Solution Type 1 (Advanced BioMatrix Inc., Poway, CA), SYBR Green PCR Master Mix (Roche) were used.

Cell culture: Human U251 and a human glioma stem cell line (gift from S. Weiss Lab) were used in these studies. The U251 and U251 -derived cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1 % penicillin/streptomycin and 1 % fungizone. The additional supplement of G418 was used for DRR-over-expressing cell lines. Cells were maintained at 37°C in a 5% C0₂ humidified atmosphere. The human glioma stem cells (hGSCs) were cultured in NeuroCult™ Proliferation Media (StemCell Technologies, Vancouver, BC) supplemented with 10% Neurocult, 1 : 1000 heparin sulfate, 20ng/ml_ hFGF2, and 20ng/ml_ hEGF. hGSCs were transfected in Neurobasal media supplemented with B27, N2, L-glutamine, 20ng/ml_ hFGF2 and 20ng/ml_ hEGF.

Western blot analysis: Cells were washed twice with cold PBS and lysed with 2% hot SDS. Proteins were separated by SDS-PAGE and transferred to nitrocellulose (BioRad). Membranes were then blocked in the appropriate blocking buffer (5% milk or 5% BSA-PBS 0.05% Tween) and incubated with indicated primary antibody overnight at 4°C. Immunoreactive bands were detected by appropriate peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., Bar Harbor, ME), and visualized by chemiluminescence (Pierce ECL solution, Thermo Scientific).

AG1478 assay: For AG1478 treatment, cells were plated in a 24 well- plate and the following day they were treated overnight with 1 -20μΜ AG1478 [θΓ 20μΜ DMSO (vehicle)], before being treated with 50ng/ml_ EGF either alone or in the presence of 1 -20μΜ AG1478 (or DMSO) for 10 minutes at 37°C.

U0126 assay: Cells were plated at a density of 80,000 cells/well in a 24-well plate. On the third day, cells were pre-treated with 5-20μΜ U0126 (or DMSO) for 2 hours at 37°C. Cells were then stimulated with EGF alone or in the presence of U0126 for 10 minutes at 37°C before lysis.

C3 transferase assay: Cells were plated at a density of 60,000 cells/well in a 24-well plate. On the third day, cells were treated with 0.5, 1 or 2ug/ml_ for 5 hours at 37°C before lysis. Cells were visualized with a Zeiss light microscope 5x objective. Cells were plated on glass coverslips and treated with C3 transferase then fixed for immuno-labeling with rhodamine phalloidin.

PP2 assay: The same protocol as the C3 transferase assay was used. On the third day, cells were treated with 5μΜ PP2 (or PP3) for 5 hours at 37°C before lysis. For the experiments in which fibronectin was used, 24- well plates were coated with 50ug/ml_ fibronectin overnight at 4°C. Fibronectin was then removed and the plate was allowed to air dry at room temperature for ~1 hr before the addition of cells. Meanwhile, cells were grown to -95% confluency in 6-well plates and pre-treated with 10μΜ PP2 for 5 hours, at which point they were then trypsinized and re-suspended in media with PP2 and plated at a cell density of 200,000 cells/well for the indicated time.

PF-228 assay: The same protocol as the C3 transferase assay was used. On the third day, cells were treated with 100nM, 500nM or 1 μΜ PF-228 for 1 hour at 37°C before lysis. For the experiments in which fibronectin was used, the same protocol was used as for PP2. Cells were pre-treated with PF-228 for 1 hour, at which point they were then trypsinized and re- suspended in media with PF-228 and plated at a cell density of 200,000 cells/well for the indicated time.

LY294002 assay: The same protocol as for U0126 was used. Cells were pre-treated with 5-20μΜ LY294002 (or DMSO) for 2 hours and then stimulated with EGF alone or in the presence of LY294002 for 10 minutes at 37°C before lysis. For the experiments in which fibronectin was used, the same protocol was used as for PP2. Cells were pre-treated with 5μΜ

LY294002 for 2 hours, at which point they were then trypsinized and re- suspended in media with LY294002 and plated at a cell density of 200,000 cells/well for the indicated time.

RGD assay: Plates were coated with fibronectin. Cells were treated with 500μΜ RGD in suspension on a rotating platform at room temperature for 30 minutes. 200,000 cells/well were plated onto fibronectin for 30 minutes. Cells that did not adhere were removed and lysed.

Immunocytochemistry: Cells were grown to -70% confluency on glass coverslips uncoated or coated with fibronectin (50μ/ιηΙ_) fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 in 0.5% PBS- BSA. Cells are then labelled with indicated primary and secondary antibodies and coverslips were mounted with Dako mounting medium. Stained cells were imaged with the Zeiss LSM700 confocal microscope using a 63x oil immersion objective. Transient transfections: Cells were plated at the appropriate confluency and transfected the following day. Either GeneJuice® (Novagen) or Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's protocol.

3D invasion assay: 25,000 cells/drop were plated onto the lid of a

10cm petri dish and hung to allow spheroid formation. On the third day, spheroids were transferred to 2% agar and on the sixth day, spheroids were implanted into a collagen type I matrix. Collagen was allowed to polymerize for 30 minutes at 37°C before the appropriate media was added (regular DMEM or supplemented with PP2/LY294002/AG1478). Spheroids were imaged over 24 hour intervals with a Zeiss light microscope 5x objective.

Animal work: All animal procedures were approved by the

Institution's Animal Care Committee and performed according to the guidelines of the Canadian Council of Animal Care.

Mouse intracerebral tumour implantation: Female CD1 athymic nude mice (Charles River, Canada) were anesthetized at six weeks of age using intra-peritoneal injection containing Ketamine, Xylazine, and

Acepromazine. The mice were placed on a stereotaxic apparatus and a midline scalp incision was made. A burrhole (3-5 mm) was created 2.2 mm lateral to the bregma using a high-powered drill. The injection needle containing 100,000 cells pretreated with the anti-sense oligonucleotide was then lowered into the burr-hole to a depth of 3.0 mm to allow tumour implantation at the center of the caudate nucleus. Animals were euthanized 3 weeks post-implantation, and their brains were harvested following

PBS/formalin animal perfusion.

Formalin Fixation, Tissue Processing & sectioning: Harvested brain specimens were placed in 10% neutral buffered formalin (Surgipath) for 72 hours at room temperature immediately following animal sacrifice. The specimens were then incubated in 70% ethanol for 24 hours at 4°C.

Following formalin fixation, the brain specimens were processed and paraffin embedded. Then, 5 μιη tissue sections were prepared using a microtome, and were mounted on a poly-L-lysine-coated glass slides (Fischer Scientific). Antigen Retrieval & Immunofluorescence: Prior to immunostaining; samples were baked in a standard laboratory oven at 60°C for 1 hour, then de-paraffinized and re-hydrated using a graded series of xylene and ethanol, respectively. Antigen retrieval was done using citrate buffer (pH 6.0) and pressure cooking for 10 minutes. The slides were then blocked for 40 minutes with a commercial protein block (Spring Bioscience), incubated for 1 hour with anti-human Sox2 primary antibody (R&D), and 20 minutes with secondary antibody (Invitrogen). Then, the slides were washed with 0.05% TBS-Tween for 15 minutes. A wash was carried out between each step throughout the staining process. After the slides were mounted, they were imaged with a LSM 700 confocal microscope using a 20x objective.

Statistical analysis: Each experiment was performed at least three times (n=3) from independent biological replicates. Data was analyzed with the statistical software GraphPad Prism 5 and has been presented as mean ± standard error of mean (SEM). p<0.05 was considered statistically significant.

DRR Induces Akt Phosphorylation To determine if DRR affects Akt activation, we assessed pAkt levels in

U251 brain cancer cells with a gain of DRR expression (DRRov, Fig. 29a) compared to parental cells (CTL) in a serum free condition. Gain of DRR expression led to significantly elevated Akt phosphorylation at both the Thr308 and Ser473 sites, while total Akt expression was similar (Fig. 29b). pAkt immunolabeling also revealed significantly elevated pAkt expression in DRRov cells compared to CTL cells (Fig. 29c).

We also assessed pAkt levels in brain cancer stem cells, which endogenously express high levels of DRR. We found high pAkt expression, which was significantly reduced upon DRR depletion using antisense oligonucleotides (Fig. 29d).

DRR Induced Akt Phosphorylation is Independent of EGFR Signaling Because EGFR signaling is amplified in 45-55% of GBMs and is a well-characterized activator of the PI3K/Akt pathway under normal and pathophysiological conditions (Akhavan et al., Neuro. Oncol. 12: 882-889, 2010; Fan and Weiss, Curr. Top. Microbiol. Immunol. 347: 279-296, 2010), we determined EGFR/pEGFR expression in the context of DRR expression. We found that DRRov cells express significantly higher levels of total cell EGFR (Fig. 30a) and cell surface EGFR compared to CTL cells (Fig. 30b). This elevated EGFR expression translated into pEGFR following EGF stimulation (Fig. 30a).

We tested the EGFR kinase inhibitor AG1478 to determine if the increased EGFR/pEGFR expression was responsible for the DRR-induced Akt phosphorylation. Surprisingly, EGFR blockade did not reduce pAkt levels in DRRov cells (Fig. 30c). And, in a 3D invasion assay, EGFR inhibition did not reduce DRRov cell invasion (Fig. 30d, e). Thus, although EGFR/pEGFR expression was elevated DRRov cells, it was not involved in DRR-induced Akt phosphorylation or cell invasion.

DRR Induced Akt Phosphorylation is Adhesion and SFK Dependent Activating inputs leading to Akt phosphorylation include

Ras/Raf/MEK/Erk, Rho-GTPases, integrin like kinase (ILK), src-family kinases (SFKs), focal adhesion kinase (FAK) and PI3K. We tested each of these inputs in order to determine how DRR regulates AKT phosphorylation.

The MEK inhibitor U0126 is a non-ATP competitive inhibitor that targets MEK1 and MEK2 and effectively inhibits downstream Erk

phosphorylation. We found that MEK inhibition did not reduce pAkt levels in DRRov cells (Fig. 31 a).

Rho-family GTPases are involved in Akt regulation and also act downstream of Akt to regulate cytoskeletal dynamics and cell movement (Vanhaesebroeck et al., Nat. Rev. Mol. Cell. Biol. 11 : 329-341 , 2010;

Wolfrum et al., Arterioscler. Thromb. Vase. Biol. 24: 1842-1847, 2004;

Higuchi et al., Curr. Biol. 11 : 1958-1962, 2001 ). Application of the Rho A, B and C inhibitor, exoenzyme C3 transferase, to DRRov generates predicted changes in cytoskeletal architecture and cell morphology including reduced stress fibers and a collapsed cell structure. Treatment with this Rho inhibitor did not affect pAKT levels in DRRov cells (Fig. 31 b).

Integrin-linked kinase (ILK) functions between integrins and RTKs and is an important activator of Akt (Hannigan et al., Nat. Rev. Cancer, 5: 51 -63, 2005; Legate et al., Nat. Rev. Mol. Cell. Biol. 7: 20-31 , 2006). We abolished ILK expression using siRNA oligonucleotides (Fig. 31 c). Application of this ILK targeting siRNA did not decrease pAKT levels in DRRov cells (Fig. 31 c).

SFKs are well-characterized effectors of integrin signaling and are key regulators of focal adhesion (FA) dynamics (Parsons and Parsons,

Oncogene 23: 7906-7909, 2004). Application of the SFK inhibitor PP2 (5 μΜ) to DRRov cells reduced pAkt levels, whereas the inactive analogue PP3 had no effect on pAkt levels (Fig. 31 d). When cells were grown on the integrin ligand fibronectin, to generate physiologic FAs, double the concentration of PP2 (10 μΜ) was necessary to reduce pAkt levels (Fig. 31 e), suggesting that SFKs are regulating DRR induced pAKT in an adhesion-dependent manner.

Activation of focal adhesion kinase (FAK) is an early event following integrin activation, is dependent upon SFKs (Parsons and Parsons,

Oncogene 23: 7906-7909, 2004), and leads to Akt phosphorylation. Using a time course analysis, we found that Akt was phosphorylated prior to FAK phosphorylation at Y397 in DRRov cells (Fig. 31 e), suggesting that FAK is not involved in DRR induced Akt phosphorylation. We directly tested this by inhibiting FAK with PF-228. Treatment of DRRov cells with 200 nM PF-228 for 1 hour prior to plating on fibronectin effectively prevented phosphorylation of FAK at Y473, yet pAkt levels were not reduced (Fig. 31 f). Interestingly, pFAK levels were lower in DRRov cells compared to CTL cells while total FAK levels are similar.

PI3K involvement was assessed by applying the inhibitor LY294002. We tested concentrations of LY294002 at 5, 10 and 20μΜ for pre-treatment times of 2, 12 and 24 hours and found that while Akt phosphorylation was effectively inhibited in CTL cells after 2 hours, LY294002 did not prevent Akt phosphorylation in DRRov cells. (Fig. 31 g). Because we found that SFK block of AKT phosphorylation was adhesion-dependent, we also tested PI3K inhibition in an adhesion-dependent manner. While 20 μΜ LY294002 was not sufficient to reduce pAKT levels in a non-adhesion dependent manner, we found that only 5μΜ LY294002 was requried to decrease Akt phosphorylation of DRRov cells grown on fibronectin (Fig. 31 h).

Together, these findings suggest that DRR induced SFK and PI3K dependent Akt phosphorylation is adhesion dependent. Integrins interact with the extracellular-matrix (ECM) through the RGD (arginine-glycine-aspartic acid) amino acid sequence. We used a modified RGD peptide that competes with the RGD domain of the ECM for integrin binding to directly test the adhesion dependence we have observed. We treated cells in suspension with the modified RGD peptide for 30 minutes prior to plating on fibronectin for 30 minutes. This treatment gave rise to an adherent and a non-adherent population of cells. In each of these cells populations there was a loss of Akt phosphorylation (Fig. 32a, b). Similarly, Akt was not phosphorylated in cells that were maintained in suspension (Fig. 32a, b).

DRR recruits Akt to Focal Adhesions

Within the cytoplasm, DRR is localized at FAs and along stress fibers (Fig. 29a). Our results showing that DRR regulates Akt phosphorylation in an adhesion-and SFK dependent manner suggests that DRR may be recruiting Akt to FAs. We tested this hypothesis by localizing Akt and pAkt in DRRov and CTL cells grown on fibronectin. For each time point studied (30m to 6h), both Akt and pAKT shared an overlapping expression pattern with vinculin in DRRov cells (Fig. 32c). In contrast, Akt and pAkt were expressed diffusely in the cytoplasm of CTL cells (Fig. 32c).

Since both DRR and SFKs regulate FA dynamics we tested if SFKs are involved in DRR-induced Akt recruitment to FAs. DRRov cells were treated with 10μΜ PP2 prior to plating on fibronectin. While FAs containing vinculin developed under these conditions, Akt was not recruited to FAs (Fig. 32d). To further explore the notion that DRR recruits Akt to FAs we utilized a mutant form of DRR, DRR^APEPE, that does not localize to FAs. We found when this form of DRR was expressed Akt was not recruited to FAs (Fig. 32e), and pAkt levels were not elevated (Fig. 32f). These experiments show that DRR, which is newly expressed in invasive gliomas, recruited Akt to FAs in an adhesion and SFK dependent manner.

SFK and PI3K Inhibitors Prevent DRR Induced Invasion

We tested if SFK and/or PI3K blockade could prevent DRRov cells from invading in a 3D invasion assay. Treatment of DRRov tumor spheroids was reduced by greater than 22% following treatment with the SFK blocker PP2 (Fig. 33a, b) and by about 66% following treatment with the PI3K blocker LY294002 (Fig. 33a, b). Combined treatment with LY294002 and PP2 led to a 16% reduction in invasion (Fig. 33d, e). CTL tumor spheroid invasion was not significantly reduced following treatment with either SFK or PI3K inhibitors (Fig. 33a, c). These experiments show that inhibition of SFKs, and to a much lesser extent PI3K, prevented Akt phosphorylation.

Antisense Oligonucleotide mediated DRR Ablation Prevents Invasion in a Mouse Model

We have shown that DRR regulates Akt phosphorylation to drive invasion, and that treatment with SFK and PI3K inhibitors can significantly reduce this invasion. We wanted to test if reduction of DRR expression could prevent brain cancer invasion. Since direct application of antisense oligonucleotides (AOs) to brain cancers is an approved and effective treatment modality, we developed AOs targeting DRR that effectively eliminate DRR expression (Fig. 34a) and assessed brain cancer invasion into normal brain. Brain cancer stem cells treated with cy5-conjugated control AOs and AOs targeting DRR were implanted in the subcallosal brain region of mice. The stem cell marker Sox was used to identify all implanted cells while cy5 labeling was used to identify tumor cells that had been treated with AOs. Whereas cancer cells treated with Cy5-conjgated control AOs readily invaded the peritumoral brain (Fig. 34b, upper panels), cancer cells treated with Cy5-conjugated AOs targeting DRR did not invade normal brain, and remained within the well-circumscribed tumor mass (Fig. 34b, lower panels). These experiments show that reduction of DRR expression using AOs targeting DRR prevented invasion in a xenograft mouse model. In sum, our findings support a model in which DRR recruits Akt to FAs in a SFK dependent manner. Akt is phosphorylated at FAs and this elevated level of pAkt remains detectable for at least 6 hours, suggesting that this recruitment generates a constitutively active form of Akt. The results support a novel mode of Akt activation following its recruitment to a specialized membrane complex.

Akt is activated in the large majority of GBMs, and emerging evidence suggests that Akt plays a role in GBM invasion (Molina et al., Neoplasia, 12: 453-463, 2010). Since Akt is not frequently mutated in GBM, its activation is presumed to be under the control of upstream RTKs. Our findings that Akt phosphorylation is not under the control of EGFR in DRR positive GBMs may provide an explanation for the negative results that EGFR inhibitors have yielded in clinical trials. Similarly, our results suggest that DRR may prove to be a useful biomarker for EGFR independent brain cancer invasion.

Antisense oligonucleotides (AOs) delivered to the resection cavity of GBM patients via implanted catheters, thereby bypassing the need for systemic delivery, is a globally approved treatment modality. We have developed an AO that targets DRR expression and thereby inhibits brain cancer invasion in a xenograft model. Upon tumor resection, catheters can be placed directly into the resection cavity providing a simple mechanism for routine AO delivery. Since GBM invasion into normal brain is a local event in over 80% or more of patients (data not shown), delivery of AO targeting invasion directly into the resection will allow for treatment to the most relevant area with minimized toxicity.

DRR Expression is Upregulated in Invasive Tumour Samples

As shown above, DRR was highly expressed in peripheral edges of glioma tumour mass. Its expression pattern within a tumour sample correlated with its pro-invasive phenotype observed in vivo and in vitro. We tested whether increased DRR expression was also correlated with an invasive phenotype in other tumour types. Analysis of various malignant tumour samples (primary site and/or correlated metastasis site) along with their corresponding normal tissues revealed that DRR is commonly expressed in normal and malignant tissue (Fig. 35). Interestingly, in almost all breast, prostate and squamous cell carcinoma samples analyzed, DRR mRNA was preferentially expressed in metastasized samples compared to primary site samples. These results indicate a correlation between highly invasive malignant cells and DRR expression in a range of tumour types, including breast, prostate and squamous cell carcinoma.

For experiments shown in Fig. 35, RNA was purified from samples according to Qiagen DNA/RNAasy™ kit instructions. cDNA synthesis was performed as follows: cDNA first strand synthesis was performed from purified RNA by mixing ~5ug RNA, oligo dT primers and dNTP together and incubating at 60 °C for 5 minutes. The reaction was cooled on ice and a prepared master mix of reverse transcriptase (RT) buffer, MgCI₂, DTT, and RNase inhibitor was added. The reaction was then incubated at 42 °C for 2 minutes and superscript II or III was added for 50 minutes at 42 °C. The reaction was terminated at 70 °C for 10 minutes, after which RNase was added and the reaction was incubated at 37 °C for 20 minutes. cDNA was stored at -80 °C until further use. Quantitative real time PCR (qRT-PCR) experiments were performed on a Bio-Rad CFX96. Primers (200 nM) and 1 ul of cDNA were mixed with Ssofast EvaGreen™ (Bio-Rad) and run at cycles of 95 °C, 2", 95 °C, 5', 60 ^oC, 5' (two step cycle, 40 cycles) and melt curve (95 °C, 5", gradient). Normalized expression of DRR was calculated by taking the relative quantity (DRR) divided by the relative quantity of a reference gene (HS14). Results were graphed as fold change expression in Fig. 35. DRR Antisense Oligonucleotides Reduce DRR Expression and Block Cell Migration and Invasion

Several DRR antisense oligonucleotides (AONs) (G5 and G6) as well as a non-targeting antisense oligonucleotide (G1 ) were tested for their ability to reduce DRR expression at the protein level. G5 is different from G6 AON due to the incorporation of FANA modification, which produces a more stable AON (Fig. 36; Table 1 ). First we tested efficacy of AONs to reduce DRR at the protein level. DRR+ cells were transfected with each AON. 72h post- transfection, cells were lysed and DRR expression level was analyzed by Western blot (Fig. 36). Both G5 and G6 AONs at a concentration of 20nM significantly decreased DRR expression levels relative to the control antisense, G1 .

As reported above, DRR reduction induces changes in cell

morphology and focal adhesion; cells are more round with an increase in cortical actin and larger focal adhesion (see also Le et al., Oncogene, 29: 4636-47, 2010). Therefore, following 72h post-transfection with the indicated antisense, DRR+ cells were immunolabeled with anti-vinculin and rhodamine phalloidin to visualize focal adhesion and actin, respectively (Fig. 37). Indeed, reduction of DRR expression level induced cells to round-up with significantly larger focal adhesion in contrast to control non-targeting antisense, where cells were more elongated with smaller focal adhesions.

To better assess AON uptake and effects, all AONs, G1 , G5 and G6, were tagged with Cy5 fluorophore. We also made a new overexpressed DRR cell line, named dsredDRR. DsredDRR cells were made by transfecting a human glioma cell line (U251 ) with a dsred-DRR plasmid, and sorting cells overexpressing dsredDRR using flow cytometry (FACS) to obtain a homogeneous population. DRR AONs untagged or Cy5 tagged, as well as Cy5-tagged control non-targeting AON (G1 ), were tested on DsRed DRR- overexpressing stable cell lines. 72h post-transfection, cells were lysed and analyzed with 12% SDS-PAGE followed by Western blot with either anti-DRR or anti-dsred antibodies. Similar to the results previously shown in Fig. 36, untagged or Cy5-tagged G5 or G6 antisense showed a reduction in DRR expression levels, as observed with anti-DRR antibody or with anti-dsred antibody relative to control antisense or untransfected cells (Fig. 38).

Therefore, cy5 addition to AONs did not affect their efficacy in reducing DRR expression.

I l l To assess changes in focal adhesion size induced by a decrease in DRR expression, DsredDRR cells were transfected with Cy5-tagged AONs. At 72h post-transfection, cells were fixed and labeled with the focal adhesion marker vinculin. Dsred cells and Cy5-AONs were directly detected in the red or blue channel, respectively (Fig. 39). From this experiment, we

demonstrated that more than 85% of the cells took up the antisense. In addition, reduction of dsredDRR (undetectable red cells) associated with bigger Fas was observed in the dsredDRR overexpressing cell line expressing cy5-G5 or cy5-G6, as compared to dsredDRR cells expressing Cy5-G1 , which did not showed reduced dsredDRR levels and exhibited small focal adhesion size (Fig. 39).

To evaluate DRR AONs' functional ability to block cell migration, we performed an in vitro scratch assay where DRR+ cells were first transfected with DRR antisense. At 72h post-transfection, a scratch was made and cells were imaged at time Oh, 24h and 48h (Fig. 40). As shown, the distance of the scratch at time zero of all conditions was similar, however within 48 hour, the scratch in control DRR+ cells could no longer be seen, while the distance of the scratch in DRR+ cells expressing the antisense (G5 or G6) was still wide open. Quantification of results demonstrated that a decrease in DRR expression levels impaired cell migration (Fig. 40).

Furthermore, to examine DRR AONs' ability to inhibit invasion, we used a 3D invasion assay where DRR+ cells were first transfected with Cy5- tagged G1 or G6 and tumour spheroids from these transfected cells were assessed in a 3D invasion assay. As shown in Fig. 41 , reduction in DRR expression levels was observed in G6 or Cy5-tagged G6 transfected DRR+ cells, in comparison with transfected Cy5-G1 or untransfected DRR+ cells. Cy5-tagged antisense expressing cells are shown in blue and invasion of a mixed population of unexpressed and expressed antisense cells are shown at day 1 , day 2 (Fig. 41 B) or at day 6 (Fig. 41 B). At day 6, G1 -cy5 expressing cells were clearly invading far from the sphere, as opposed to G6-cy5 expressing cells (Fig. 41 B). Indeed, quantitative data indicated that reduction of DRR expression leads to a decrease in cell invasion (Fig. 41 C). Cancer stem cells (CSCs) represent a subpopulation of cells that can escape chemotherapy. Therapeutic drugs that effectively target CSCs would be beneficial for cancer treatments. Therefore, we investigated the efficacy of DRR AONs on a human glioma stem cell line (hGSC). As observed in Fig. 42, hGSCs expressing DRR targeted AONs (G5 or G6) showed decreased DRR expression levels in comparison with non-targeted control antisense (G1 ) or untransfected cells. Notably, in a 3D invasion assay, we showed that both G5 and G6 AONs successfully inhibited hGSC invasion (Fig. 42).

To further assess brain cancer invasion into normal brain, hGSC cells were first transfected with control non-targeting antisense (G1 -cy5) or with targeted AONs (G5-cy5 or G6-cy5) and 72h post-transfection, cells were implanted in the subcallosal brain region of mice. Sections of mice brain are shown in Fig. 43 in which cy5 labeling was used to identify tumor cells that had been treated with AONs and H&E stained sections are shown to identify the tumor mass. With G1 transfection, significant cy5-G1 expressing cells were invading the peritumoral brain (upper panels), whereas with G5 and G6 transfection, cy5 labelled cells remained within circumscribed tumor mass area (lower panels). These results indicate that DRR AONs could be transfected in human glioma stem cells and induced changes in the cells that reduce their capacity to invade.

DRR siRNAs Reduce DRR Expression and Induce Changes in Cell Morphology

In order to obtain more efficient reduction of DRR expression levels, 3 DRR siRNAs were designed, as well as a non-targeting siRNA as control (Fig. 44). To evaluate the efficacy of these siRNAs, DRR+ cells were transfected with each DRR siRNA. 72h post-transfection, cells were lysed and DRR expression levels were analyzed by Western blot (Fig. 44). As seen in Fig. 44, the most potent DRR siRNA was siRNAI (also referred to as DRR1 siRNA), where DRR expression level was minimal compared to non- targeting DRR siRNA (DRR4). As reported above, DRR reduction induced changes in cell

morphology and focal adhesion; cells became more round with more cortical actin with larger focal adhesions (see also Le et al., Oncogene, 29: 4636-47, 2010). Therefore, DRR+ cells were transfected with non-targeting siRNA or with DRR siRNAI (also referred to as DRR1 siRNA) and immunolabeled with rhodamine phalloidin and anti-vinculin to visualize actin and focal adhesion (Fig. 45). Indeed, reduction of DRR expression level induced cells to roundup with significantly larger focal adhesion in contrast with control non- targeting siRNA, where DRR+ cells were more elongated. Furthermore, to image DRR siRNA uptake and effect in cells, DRR siRNA-2 (also referred to as DRR2 siRNA) was conjugated to Cy-5 and, following DRR+ cell transfection with DRR siRNA-2Cy5, cells were labelled with rhodamine- phalloidin and anti-vinculin (Fig. 46). Again, we observed changes in cell morphology and bigger focal adhesion with reduced DRR expression.

The Materials and Methods for experiments and results shown in

Figures 36-46 are as follows:

DRR antisense oligonucleotide synthesis and purification:

Antisense oligonucleotides (G5, Cy5-G5, G6, and Cy5-G6) were designed to target DRR mRNA (NM_007177) in the open reading frame at position 576- 595. G1 and Cy5-G1 are non-targeting negative control sequences. All antisense oligonucleotides were synthesized on an ABI 3400 DNA

synthesizer from Applied Biosystems. Syntheses were performed at a 1 μιηοΐβ scale. Unylink CPG (ChemGenes) was used as a solid support. DNA phosphoramidites were prepared as 0.1 M solutions in dry acetonitrile (ACN). 2'-deoxy-2'-fluoro-B-D-arabinose nucleic acid (2'F-ANA) phosphoramidites were prepared as 0.12M solutions in dry ACN. Detritylations (1 10s) used 3% trichloroacetic acid in dichloromethane. Amidite couplings were activated with 5-Ethylthiotetrazole (0.25M in ACN, ChemGenes). Coupling times were 600s for DNA and 2'F-ANA phosphoramidites; 900s for guanosine couplings.

Sulfurizations were accomplished with 0.1 M xanthane hydride in 1 : 1 v/v dry pyridine/ACN for 2.5 minutes, with an addition of fresh reagent after 1 .25 min. Capping was performed with acetic anhydride in tetrahydrofuran (THF), and 16% /V-methylimidazole in THF. Dried CPG was transferred from the column to a screw-cap eppendorf, and deprotection and cleavage from the solid support was effected using 1 ml_ of 3: 1 v/v aqueous NH OH:EtOH for 48 hours at room temperature. Samples were centrifuged, and the deprotection solution was decanted away from the solid support. Samples were vented for 2.5 hours, chilled on dry ice, and the deprotection solution was removed in a speedvac lyophilizer.

Oligonucleotides were purified by reverse phase HPLC using a Waters 1525 HPLC and a Varian Pursuit 5 semi-preparative reverse phase C18 column. A stationary phase of 100mM triethylammonium acetate in water with 5% ACN (pH 7) and a mobile phase of HPLC-grade ACN was used. Purifications employed a gradient of 5%-35% ACN over 35min. Purified samples were lypophilized to dryness from water 3 times. All oligonucleotides were quantitated by UV absorbance on a Cary 300 UV, using extinction coefficients calculated used the online IDT OligoAnalyzer tool

(http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). 2'F-ANA- modified AONs were treated as DNA for extinction coefficient calculations. Oligonucleotides were characterized by LC-MS on a Waters Q-TOF2 using an ESI NanoSpray source. A CapLC (Waters) with a C18 trap column was used for LC prior to injections.

Cell culture and transfection: DRR+ and dsRed-DRR expressing cells were cultured as described (see Le et al., Oncogene, 29: 4636-47, 2010). 46EF stem cells were kindly provided by Dr. Samuel Weiss (University of Calgary). 46EF stem cells were isolated as previously performed (Kelly et al., Stem Cells 27: 1722-33, 2009) and expanded in neurosphere cultures. Spheres were cultured in complete Neurocult-NS-A proliferation Medium (Neurocult basal medium containing: Neurocult NS-A differentiation supplement at a concentration of 1/10 dilution, 20 ng/ml rh EGF, 20 ng/ml rh bFGF and 2μg/ml Heparin) from stem cells technologies. When spheres appeared large enough for passaging (<300 μιη in diameter), they were collected in a tube and spun at 1200rpm for 3 minutes. To dissociate the spheres, 700ul of Accumax (Millipore) was added to the cell pellet and incubated for 5 minutes at 37°C, they were then washed with PBS, centhfuged and resuspended in complete Neurocult-NS-A proliferation medium and seeded at a concentration of 200 000 cells/flask.

One day before transfection, DRR+ and dsRed-DRR cells were plated in 24 well or 6 well plates such that they reached 75% confluency at the time of transfection. 46 EF stem cells were plated at 400 000 cells/well in a 6 well plate and were transfected on the same day. All transfection was done using lipofectamine 2000 reagent according to the manufacturer's indications. Briefly, lipofectamine was gently mixed in opti-MEM and left at room temperature for 5min. DRR oligomer was first mixed with opti-MEM (such that the final concentration added to the cells was 20nM) and gently mixed with lipofectamine. Lipofectamine-DRR oligomer complexes were incubated at room temperature for 20 minutes before being added to the cells. The day after transfection, fresh cell media was added to the transfected cells. Cells were fixed or lysed following 72h post-transfection.

Western blot analysis: Cells were lysed with 2% hot SDS and separated by 12% SDS-PAGE and transferred to nitrocellulose before being analysed by western blot using a specific rabbit anti-DRR antibody.

Immunoreactive bands were detected by a goat anti-rabbit antibody linked to horseradish peroxidase (Jackson), and visualized by chemiluminescence (super ECL, Pierce).

Immunofluorescence: Cells were fixed with 4% PFA and

permeabilized with 0.5% TritonX-100 before being processed for

immunostaining. Cells were labelled with mouse anti-vinculin to visualize focal adhesions and rhodamine-phalloidin was used to stain actin.

Fluorescently labelled cells were visualized with a Zeiss LSM700 confocal microscope using 63x objectives.

Scratch assay: Following 72h post-transfection, cells reached a monolayer. We used a 200μΙ pipette to perform a scratch. Cells were rinsed 3 times with PBS and fresh media was added to the cells. Images were captured with a 5x objective at the beginning of the scratch and at 24h and 48h. For each image, distances between one side of the scratch and the other were measured. The distance (μιη) of cell migration was quantified by measuring the distance of the scratch at each time interval and subtracting it from the distance of the scratch at time zero.

3D-lnvasion assay: We plated cell drops in a cover of 10 cm culture dish (25,000 cells/drop (20 μΙ)). Tumor spheroids were generated using the hanging drop method and implanted in a collagen type 1 matrix as previously described (Werbowetski-Ogilvie et al., Cancer Research 66: 1464-1472, 2006). The implanted spheroids were imaged after the following time points (0, 24, 48 and 72 hours). Invading areas were measured by calculating the extreme diameter at 4 different angles and by subtracting the extreme diameter of the spheroids at time zero.

Mouse intracerebral tumor implantation: Female CD1 athymic nude mice (Charles River, Canada) were anesthetized at six weeks of age using intraperitoneal injection containing Ketamine, Xylazine, and

Acepromazine. The mice were placed on a stereotaxic apparatus and a midline scalp incision was made. A burr-hole (3-5 mm) was created 2.2 mm lateral to the bregma using a high-powered drill. The injection needle containing 100,000 cells 72h post-transfected with cy5-taged antisense was then lowered into the burr-hole to a depth of 3.0 mm to allow tumor implantation at the center of the caudate nucleus. Animals were euthanized 3 weeks post-implantation, and their brains were harvested following

PBS/formalin animal perfusion. All animal procedures were approved by the Institution's Animal Care Committee and performed according to the guidelines of the Canadian Council of Animal Care.

Formalin Fixation, Tissue Processing & sectioning: Harvested brain specimens were placed in 10% neutral buffered formalin (Surgipath) for 72 hours at room temperature immediately following animal sacrifice. The specimens were then incubated in 70% ethanol for 24 hours at 4 degrees. Following formalin fixation, the brain specimens were taken to the tissue processor for dehydration and tissue infiltration with paraffin at 60 degrees. Finally, the processed brains were embedded in paraffin blocks for tissue sectioning. After paraffin processing and embedding, 5 urn tissue sections were prepared using a microtome, and were mounted on a poly-L-lysine- coated glass slides (Fisher Scientific).

Antigen Retrieval & Immunofluorescence: Prior to immunostaining; samples were baked in a standard laboratory oven at 60 °C for 1 hour, then deparaffinized and rehydrated using a graded series of xylene and ethanol, respectively. Antigen retrieval was done using citrate buffer (pH 6.0) and pressure cooking for 10 minutes. The slides were then blocked for 40 minutes with a commercial protein block (Spring Bioscience), incubated for 1 hour with anti-human Sox-2 antibody (R&D), and 20 minutes with Alexa-567 conjugated secondary antibody (Invitrogen). Washing with 0.05% TBS- Tween for 15 minutes was carried out between each step throughout the staining process. After the slides were mounted, they were imaged using the LSM 700 confocal microscope.

The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS: 1 . A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing an antisense RNA comprising the sequence of SEQ ID NOs: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16, or a fragment or derivative thereof, to tumor cells, wherein the antisense RNA reduces the expression of DRR in the tumor cells.

2. An antisense RNA for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising the sequence of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16, or a fragment or derivative thereof.

3. A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing to tumor cells a DNA molecule comprising a sequence which encodes the sequence of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16 or a fragment or derivative thereof, wherein the DNA encodes an antisense RNA suitable for reducing the expression of DRR in the tumor cells.

4. The method of claim 3, wherein the DNA molecule is inserted in an expression vector suitable for production of antisense RNA. 5. An expression vector comprising a sequence encoding for the sequence of SEQ ID NO: 2,

5, 6, 7, 8, 9, 10, 14, 15 or 16, or a fragment or derivative thereof.

6. A method of treating cancer comprising administering the antisense RNA of claim 2 or a vector that encodes the antisense RNA of claim 2 to a subject in need thereof.

7. A method of delaying progression of cancer comprising administering the antisense RNA of claim 2 or a vector that encodes the antisense RNA of claim 2 to a subject in need thereof.

8. The method of claim 6 or 7, wherein the antisense RNA and/or the vector are used in combination with one or more cancer therapies selected from the group consisting of surgical resection, chemotherapy, radiation therapy, immunotherapy, and gene therapy.

9. The method of any one of claims 1 , 3, 4 and 6 to 8, wherein the tumor cells are glioma cells, breast carcinoma cells, prostate carcinoma cells, squamous cell carcinoma cells, lung carcinoma cells, colon carcinoma cells, or renal cell carcinoma cells.

10. The method of claim 9, wherein the glioma cells are glioblastoma cells.

1 1 . The antisense RNA of claim 2, wherein the tumor cells are glioma cells.

12. The antisense RNA of claim 1 1 , wherein the glioma cells are glioblastoma cells.

13. A pharmaceutical composition for treatment of cancer comprising the antisense RNA of claim 2 or a vector that encodes the antisense RNA of claim 2, and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein the cancer is glioma, breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma.

15. The pharmaceutical composition of claim 14, wherein the glioma is malignant glioblastoma.

16. A kit comprising the pharmaceutical composition according to any one of claims 13 to 15, and instructions for use thereof.

17. The kit of claim 16, further comprising a second active compound suitable for treating glioma and/or for delaying progression thereof, for simultaneous, separate or sequential administration to a subject.

18. A method for enhancing efficacy of a cancer therapy for treatment of glioma, comprising administering the antisense RNA of claim 2 or a vector that encodes the antisense RNA of claim 2 to a subject in need thereof, and simultaneously, separately or sequentially administrating said cancer therapy, wherein said cancer therapy is selected from the group consisting of surgical resection, chemotherapy, radiation therapy, immunotherapy, and gene therapy.

19. The method of claim 18, wherein the glioma is malignant glioblastoma.

20. A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising providing to tumor cells an antisense RNA having the sequence of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16 or a fragment or derivative thereof, wherein the antisense RNA reduces expression of DRR in the tumor cells.

21 . A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising providing to tumor cells a DNA molecule comprising the sequence of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16 or a fragment or derivative thereof, wherein the DNA encodes an antisense RNA suitable for reducing expression of DRR in the tumor cells.

22. A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising administering the antisense RNA of claim 2 or a vector that encodes the antisense RNA of claim 2 to the subject.

23. A method for diagnosis or prognosis of glioma in a subject, comprising measuring DRR expression in the glioma cells of the subject, wherein DRR expression indicates invasiveness of the cells.

24. A method for visualizing invasive glioma cells in a subject, comprising contacting glioma cells with a molecule which specifically binds DRR protein or mRNA and measuring DRR protein or mRNA levels in the cells, wherein cells which express DRR are invasive.

25. A kit for diagnosis or prognosis of invasive glioma in a subject, comprising a detectably-labelled probe specific for DRR RNA or protein, a reporter means for detecting binding of the probe to the DRR RNA or protein, and instructions for use thereof.

26. A method for treating cancer comprising administering a therapeutic RNA which reduces the expression of DRR, or a DNA or vector encoding the therapeutic RNA, to a subject in need thereof.

27. The method of claim 27, wherein progression of the cancer is delayed.

28. The method of claim 26 or 27, wherein malignant cell invasion is inhibited.

29. The method of claim 28, wherein malignant glial cell invasion is inhibited.

30. The method of any one of claims 26 to 29, wherein the cancer is glioma, preferably malignany glioma, and more preferably glioblastoma, or wherein the cancer is breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma.

31 . The method of any one of claims 26 to 30, wherein the therapeutic RNA is an antisense RNA.

32. The method of any one of claims 26 to 31 , wherein the therapeutic RNA is complementary to or specifically hybridizes to DRR mRNA or a fragment or derivative thereof.

33. A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing a therapeutic RNA complementary to or specifically hybridizing to DRR mRNA to the tumor cells, wherein the therapeutic RNA reduces the expression of DRR in the tumor cells.

34. The method of claim 33, wherein the therapeutic RNA comprises an antisense RNA.

35. The method of claim 33 or 34, wherein the therapeutic RNA comprises an antisense RNA which has the structure of an altimer, a gapmer or an aptamer, and/or comprises a modified nucleoside or nucleotide.

36. The method of claim 35, wherein the modified nucleoside or nucleotide is FANA.

37. An RNA having the sequence set forth in SEQ ID NOs: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16.

38. A DNA encoding an RNA having the sequence set forth in SEQ ID NOs: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16.

39. A DNA having the sequence of the RNA set forth in SEQ ID NOs: 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16.

40. A pharmaceutical composition comprising the RNA or DNA of any one of claims 37 to 39 and a pharmaceutically acceptable carrier.

41 . A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing a siRNA comprising the sequence of SEQ ID NO: 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, to tumor cells, wherein the siRNA reduces the expression of DRR in the tumor cells.

42. A siRNA for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising the sequence of SEQ ID NO: 17/18, 19/20, 21 /22 or 23/24, or a fragment or derivative thereof.

43. A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing to tumor cells a DNA molecule comprising a sequence which encodes the sequence of SEQ ID NO: 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, wherein the DNA encodes a siRNA suitable for reducing the expression of DRR in the tumor cells.

44. The method of claim 43, wherein the DNA molecule is inserted in an expression vector suitable for production of siRNA.

45. An expression vector comprising a sequence encoding the sequence of SEQ ID NO: 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof.

46. A method of treating cancer comprising administering the siRNA of claim 42 or a vector that encodes the siRNA of claim 42 to a subject in need thereof.

47. A method of delaying progression of cancer comprising administering the siRNA of claim 42 or a vector that encodes the siRNA of claim 42 to a subject in need thereof.

48. The method of claim 46 or 47, wherein the siRNA and/or the vector are used in combination with one or more cancer therapies selected from the group consisting of surgical resection, chemotherapy, radiation therapy, immunotherapy, and gene therapy.

49. The method of any one of claims 41 , 43, 44 and 46 to 48, wherein the tumor cells are glioma cells, breast carcinoma cells, prostate carcinoma cells, squamous cell carcinoma cells, lung carcinoma cells, colon carcinoma cells, or renal cell carcinoma cells.

50. The method of claim 49, wherein the glioma cells are glioblastoma cells.

51 . The siRNA of claim 42, wherein the tumor cells are glioma cells, breast carcinoma cells, prostate carcinoma cells, squamous cell carcinoma cells, lung carcinoma cells, colon carcinoma cells, or renal cell carcinoma cells.

52. The siRNA of claim 51 , wherein the glioma cells are glioblastoma cells.

53. A pharmaceutical composition for treatment of cancer comprising the siRNA of claim 42 or a vector that encodes the siRNA of claim 42, and a pharmaceutically acceptable carrier.

54. The pharmaceutical composition of claim 53, wherein the cancer is glioma, breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma.

55. The pharmaceutical composition of claim 54, wherein the glioma is malignant glioblastoma.

56. A kit comprising the pharmaceutical composition according to any one of claims 53 to 55, and instructions for use thereof.

57. The kit of claim 56, further comprising a second active compound suitable for treating glioma and/or for delaying progression thereof, for simultaneous, separate or sequential administration to a subject.

58. A method for enhancing efficacy of a cancer therapy for treatment of glioma, comprising administering the siRNA of claim 42 or a vector that encodes the siRNA of claim 42 to a subject in need thereof, and simultaneously, separately or sequentially administrating said cancer therapy, wherein said cancer therapy is selected from the group consisting of surgical resection, chemotherapy, radiation therapy, immunotherapy, and gene therapy.

59. The method of claim 58, wherein the glioma is malignant glioblastoma.

60. A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising providing to tumor cells a siRNA having the sequence of SEQ ID NO: 17/18, 19/20, 21/22 or 23/24, or a fragment or derivative thereof, wherein the siRNA reduces expression of DRR in the tumor cells.

61 . A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising providing to tumor cells a DNA molecule comprising the sequence of SEQ ID NO: 17/18, 19/20, 21 /22 or 23/24, or a fragment or derivative thereof, wherein the DNA encodes a siRNA suitable for reducing expression of DRR in the tumor cells.

62. A method for inhibiting malignant glial cell invasion in a subject in need thereof, comprising administering the siRNA of claim 42 or a vector that encodes the siRNA of claim 42 to the subject.

63. A method for diagnosis or prognosis of glioma in a subject, comprising measuring DRR expression in glioma cells of the subject, wherein DRR expression indicates invasiveness of the cells.

64. A method for visualizing invasive glioma cells in a subject, comprising contacting glioma cells with a molecule which specifically binds DRR protein or mRNA and measuring DRR protein or mRNA levels in the cells, wherein cells which express DRR are invasive.

65. A kit for diagnosis or prognosis of invasive glioma in a subject, comprising a detectably-labelled probe specific for DRR RNA or protein, a reporter means for detecting binding of the probe to the DRR RNA or protein, and instructions for use thereof.

66. A method for treating cancer comprising administering a therapeutic RNA which reduces expression of DRR, or a DNA or vector encoding the therapeutic RNA, to a subject in need thereof.

67. The method of claim 67, wherein progression of the cancer is delayed.

68. The method of claim 66 or 67, wherein malignant cell invasion is inhibited.

69. The method of claim 68, wherein malignant glial cell invasion is inhibited.

70. The method of any one of claims 66 to 69, wherein the cancer is glioma, preferably malignany glioma, and more preferably glioblastoma, or breast carcinoma, prostate carcinoma, squamous cell carcinoma, lung carcinoma, colon carcinoma, or renal cell carcinoma.

71 . The method of any one of claims 66 to 70, wherein the therapeutic RNA is a siRNA.

72. The method of any one of claims 66 to 71 , wherein the therapeutic RNA is complementary to or specifically hybridizes to DRR mRNA or a fragment or derivative thereof.

73. A method for reducing expression of downregulated in renal cell carcinoma (DRR) in tumor cells, comprising providing a therapeutic RNA complementary to or specifically hybridizing to DRR mRNA to the tumor cells, wherein the therapeutic RNA reduces the expression of DRR in the tumor cells.

74. The method of claim 73, wherein the therapeutic RNA comprises a siRNA.

75. The method of claim 73 or 74, wherein the therapeutic RNA comprises a siRNA which comprises a modified nucleoside or nucleotide.

76. The method of claim 75, wherein the modified nucleoside or nucleotide is FANA.

77. A siRNA having the sequence set forth in SEQ ID NO: 17/18, 19/20, 21 /22 or 23/24.

78. A DNA encoding an RNA having the sequence set forth in SEQ ID NO: 17/18, 19/20, 21/22 or 23/24.

79. A DNA having the sequence of the RNA set forth in SEQ ID NO: 17/18, 19/20, 21/22 or 23/24.

80. A pharmaceutical composition comprising the RNA or DNA of any one of claims 77 to 79 and a pharmaceutically acceptable carrier.

81 . An siRNA molecule, wherein said siRNA molecule consists of: (a) a duplex region; and (b) either no overhang regions or at least one overhang region, wherein each overhang region contains six or fewer nucleotides, wherein the duplex region consists of a sense region and an antisense region, wherein said sense region and said antisense region together form said duplex region and each of said sense region and said antisense region is 18-30 nucleotides in length and said antisense region comprises a sequence that is the complement of SEQ ID NO: 4 or a fragment or portion thereof.

82. The siRNA molecule of claim 81 , wherein said antisense region and said sense region are each 19-25 nucleotides in length.

83. The siRNA molecule of claim 82, wherein said antisense region and said sense region are each 21 nucleotides in length.

84. The siRNA molecule of any one of claims 81 to 83, wherein said siRNA molecule has at least one overhang region.

85. The siRNA molecule of any one of claims 81 to 83, wherein said siRNA molecule has no overhang regions.

86. The siRNA molecule of any one of claims 81 to 85, wherein said siRNA comprises one or more FANA nucleotides.

87. The siRNA molecule of any one of claims 81 to 86, wherein said siRNA comprises one or more FRNA residues.

88. The siRNA molecule of any one of claims 81 to 87, wherein said siRNA comprises the sequence of siRNAI (SEQ ID NO: 17/18), siRNA2 (SEQ ID NO: 19/20) or siRNA3 (SEQ ID NO: 23/24).

89. The siRNA molecule of any one of claims 81 to 88, wherein said siRNA consists of siRNAI , siRNA2 or siRNA3.

90. The siRNA molecule of any one of claims 81 to 89, wherein said antisense region comprises a sequence that is the complement of nucleotides at positions 227-245 of SEQ ID NO: 4.

91 . An siRNA molecule comprising a sense region and an antisense region that downregulates expression of a DRR gene via RNA interference (RNAi), wherein the sense region comprises a nucleotide sequence set forth in SEQ ID NO: 17, 19, 21 or 23, and wherein the antisense region comprises a sequence that is complementary to a nucleotide sequence consisting of SEQ ID NO: 4 or a fragment or portion thereof.

92. The siRNA molecule of claim 91 , wherein the antisense region comprises a nucleotide sequence set forth in SEQ ID NO: 18, 20, 22 or 24.

93. The siRNA molecule of claim 91 , wherein the antisense region consists of a nucleotide sequence which is set forth in SEQ ID NO: 18, 20, 22 or 24.

94. The siRNA molecule of claim 91 , wherein the antisense region is complementary to nucleotides at positions 227-245 of SEQ ID NO: 4.

95. A recombinant nucleic acid construct or vector comprising a nucleic acid that is capable of directing transcription of a small interfering RNA

(siRNA), the nucleic acid comprising: (a) at least one promoter; (b) a DNA polynucleotide segment that is operably linked to the promoter, (c) a linker sequence comprising at least 4 nucleotides operably linked to the DNA polynucleotide segment of (b); and (d) operably linked to the linker sequence a second polynucleotide, wherein the polynucleotide segment of (b) comprises a polynucleotide that is selected from the group consisting of SEQ ID NOs: 17, 19, 21 and 23, wherein the second polynucleotide of (d) comprises a polynucleotide that is complementary to at least one polynucleotide that is selected from the group consisting of SEQ ID Nos: 17, 19, 21 and 23.

96. The recombinant nucleic acid construct or vector of claim 95, wherein the DNA polynucleotide sequence comprises SEQ ID NO: 17, 19, 21 or 23 and the second polynucleotide comprises SEQ ID NO: 18, 20, 22 or 24.

97. An isolated host cell transformed or transfected with the recombinant nucleic acid construct or vector of claim 95 or 96.

98. An siRNA expression vector for downregulating expression of DRR in a subject in need therof, wherein the vector comprises:

(1 ) a bacterial cassette comprising a bacterial origin of replication and a bacterial selectable marker M 1 ;

(2) a cassette for selection in eukaryotic cells comprising a selectable marker M2 for eukaryotic cells, and in particular for mammalian cells, under the control of an appropriate promoter;

(3) an siRNA transcription cassette comprising at least one region encoding an siRNA corresponding to a DRR gene, under control of regulatory elements for transcription in mammalian cells, which regulatory elements include at least one promoter capable of transcribing an siRNA in mammalian cells and a transcription terminator; wherein said siRNA transcription cassette is immediately downstream of the transcription initiation site or else a maximum of at most 20 base pairs away from the latter; said transcription initiation site being CCG and said siRNA transcription cassette comprising, downstream of the sequence encoding the siRNA, a transcription terminator which comprises a sequence of 6 consecutive thymidines, in the sense strand of the construct.

99. The vector of claim 98, wherein the DRR gene comprises the sequence set forth in SEQ ID NO:4 or a fragment or portion thereof.

100. The vector of claim 98 or 99, wherein the siRNA is the siRNA molecule of any one of claims 81 to 93.

101 . The vector of claim 98 or 99, wherein the siRNA comprises the sequence set forth in SEQ ID NO: 17/18, 19/20, 21/22 or 23/24.

102. The vector of claim 98 or 99, wherein the siRNA consists of the sequence set forth in SEQ ID NO: 17/18, 19/20, 21/22 or 23/24.

103. A pharmaceutical composition comprising the siRNA of any one of claims 81 to 94 or the vector of any one of claims 95-96 and 98-102, and a pharmaceutically acceptable carrier.

104. An siRNA molecule, wherein said siRNA molecule consists of a duplex region, said duplex region consisting of a sense region and an antisense region, wherein:

(a) said sense region and said antisense region together form said duplex region; (b) each of said sense region and said antisense region is 18-30 nucleotides in length; and

(c) said antisense region comprises a sequence that is complementary to a nucleotide sequence consisting of SEQ I D NO: 4 or a fragment or portion thereof.

105. The siRNA molecule of claim 104, wherein the sequence of said antisense region is complementary to a sequence comprising nucleotides from position 227 to 245 of SEQ ID NO: 4.

106. The siRNA molecule of claim 104, wherein the sequence of said antisense region is complementary to a sequence consisting of nucleotides from position 227 to 245 of SEQ ID NO: 4.

107. The siRNA molecule of any one of claims 104 to 106, wherein said antisense region and said sense region are each 19-25 nucleotides in length.

108. The siRNA molecule of any one of claims 104 to 107, wherein the sense region comprises the sequence set forth in SEQ ID NO: 17, 19, 21 or 23 and the antisense region comprises the sequence set forth in SEQ ID NO: 18, 20, 22 or 24.

109. The siRNA molecule of any one of claims 104 to 108, wherein the siRNA molecule consists of a duplex comprising the sequence set forth in SEQ I D NO: 17/18, 19/20, 21/22 or 23/24.

1 10. The siRNA molecule of any one of claims 104 to 108, wherein the siRNA molecule consists of a duplex consisting of the sequence set forth in SEQ I D NO: 17/18, 19/20, 21/22 or 23/24.

1 1 1 . The siRNA molecule of any one of the preceding claims, wherein the siRNA molecule comprises one or more FANA nucleotides and/or one or more FRNA residues.