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WO2016083466A1 - Novel tumor suppressor gene and uses thereof for the treatment and diagnosis of cancer - Google Patents

Novel tumor suppressor gene and uses thereof for the treatment and diagnosis of cancer Download PDF

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Publication number
WO2016083466A1
WO2016083466A1 PCT/EP2015/077679 EP2015077679W WO2016083466A1 WO 2016083466 A1 WO2016083466 A1 WO 2016083466A1 EP 2015077679 W EP2015077679 W EP 2015077679W WO 2016083466 A1 WO2016083466 A1 WO 2016083466A1
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Prior art keywords
malignant
carcinoma
cell
adenocarcinoma
tumor
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French (fr)
Inventor
Asma SMAHI
Arnold Munnich
Elodie BAL
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Fondation Imagine
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Fondation Imagine
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Publication of WO2016083466A1 publication Critical patent/WO2016083466A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel tumor suppressor gene and uses thereof for the treatment and diagnosis of cancer.
  • a tumor suppressor gene is a gene that protects a cell from one step on the path to cancer. When this gene mutates to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes. The loss of these genes may be even more important than proto-oncogene/oncogene activation for the formation of many kinds of human cancer cells. For instance, tumor- suppressor genes may have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. Examples of tumor suppressor genes include but are not limited to TP53, PTEN, pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14.
  • the present invention relates to a novel tumor suppressor gene and uses thereof for the treatment of cancer.
  • the present invention is defined by the claims.
  • Cutaneous basal cell carcinoma is the most common human cancer. While most cases are sporadic, some forms are inherited, as in Bazex-Dupre-Christol syndrome (BDCS), an X-linked dominant cancer-prone genodermatosis. Studying a series of 6 BDCS families, the inventors found a mutation in a gene encoding the actin-related protein Arp-Tl (ACTRTl) in 2/6 BDCS patients, resulted in absent expression of Arp-Tl in BDCS tumors. Wild-type Arp-Tl, but not its mutant counterpart could bind chromatin.
  • ACTRTl actin-related protein Arp-Tl
  • ACTRTl Overexpression of ACTRTl resulted in an increased number of cells arrested in S-phase, which supports a role for Arp-Tl in cell cycle regulation.
  • ACTRTl When stably expressed in MDA-MB231 cell line, ACTRTl reduced proliferation and migration.
  • Arp-Tl was found to act as a tumor-suppressor via its direct binding to Hedgehog signaling target genes, thus inhibiting their expression.
  • ACTRT1 has its general meaning in the art and refers to the Actin-Related Protein Tl .
  • An exemplary amino acid sequence is SEQ ID NO: l and an exemplary nucleic acid sequence is SEQ ID NO:2.
  • a first object of the present invention relates to a method which involves contacting a cancer cell with an Arp-Tl polypeptide of the invention, or a functional variant or fragment thereof, in order to inhibit proliferation and migration of the cancer cell.
  • polypeptides of the invention include all those disclosed herein. They can be, for example, bArp-Tl (SEQ ID NO: l) and functional variant or fragments of this polypeptide.
  • the polypeptides embraced by the invention also include fusion proteins that contain either full-length Arp-Tl or a functional fragment of it fused to unrelated amino acid sequence.
  • the unrelated sequences can be additional functional domains or signal peptides. Signal peptides are described in greater detail and exemplified below.
  • the polypeptides can be any of those described above but with one or more (e.g., 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11 ; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21 ; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71 ; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98;
  • the functional variant consists of a polypeptide having a sequence having at least 70% of identity with SEQ ID NO: l .
  • a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91 ; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).
  • the polypeptide of the invention is a functional conservative variant of the polypeptide.
  • the term "function-conservative variant” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • a “function-conservative variant” also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared (i.e. inhibition of migration and proliferation of cancer cells).
  • Functional properties of the polypeptide of the invention could typically be assessed in any functional assay as described in the EXAMPLE.
  • the fragment comprises at least 10; 11; 12; 13; 14; 14; 15; 16;
  • polypeptides can be purified from natural sources (e.g., any cell that naturally produces Arp-Tl polypeptides). Smaller peptides (e.g. less than 50 amino acids long) can also be conveniently synthesized by standard chemical means.
  • both polypeptides and peptides can be produced by standard in vitro recombinant DNA techniques and in vivo transgenesis, using nucleotide sequences encoding the appropriate polypeptides or peptides. Methods well-known to those skilled in the art can be used to construct expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals.
  • Polypeptides and fragments of the invention also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo.
  • a blocking agent to facilitate survival of the relevant polypeptide in vivo.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • Peptidomimetic compounds that are designed based upon the amino acid sequences of the functional peptide fragments.
  • Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide.
  • the peptide motif provides the peptidomimetic compound with the ability to inhibit the proliferation of cancer cells in a manner qualitatively identical to that of the Arp-Tl functional fragment from which the peptidomimetic was derived.
  • Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased cell permeability and prolonged biological half-life.
  • the peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based.
  • Several types of chemical bonds e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics.
  • a further aspect of the present invention relates to a method which involves contacting a cancer cell with an ACTRT1 nucleic acid molecule of the invention, in order to inhibit proliferation and migration of the cancer cell.
  • an "ACTRT1 nucleic acid molecule” is a nucleic acid molecule which encodes a hArp-Tl (e.g. SEQ ID NO: l) polypeptide as well as functional variants or fragments of this polypeptide as described above.
  • the ACTRT1 nucleic acid molecules of the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Segments of these molecules are also considered within the scope of the invention, and can be produced by, for example, the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases.
  • PCR polymerase chain reaction
  • a ribonucleic acid (RNA) molecule can be produced by in vitro transcription.
  • nucleic acid molecules of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide (for example, the polypeptides with SEQ ID NO: l).
  • these nucleic acid molecules are not limited to coding sequences, e.g., they can include some or all of the non-coding sequences that lie upstream or downstream from a coding sequence.
  • the nucleic acid molecules of the invention can be synthesized (for example, by phosphoramidite-based synthesis) or obtained from a biological cell, such as the cell of a mammal.
  • the nucleic acids can be those of a human, non-human primate (e.g., monkey), mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat. Combinations or modifications of the nucleotides within these types of nucleic acids are also encompassed.
  • the isolated nucleic acid molecules of the invention encompass segments that are not found as such in the natural state.
  • the invention encompasses recombinant nucleic acid molecules (for example, isolated nucleic acid molecules encoding hArp-Tl) incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).
  • the nucleic acid molecule of the invention is SEQ ID NO:2 or a nucleic acid sequence having at least 70% of identity with SEQ ID NO:2.
  • the invention also encompasses: (a) vectors that contain any of the foregoing ACTRT1 nucleic acid molecules; (b) expression vectors that contain any of the foregoing ACTRT1 nucleic acid molecules operably linked to any transcriptional/translational regulatory elements (examples of which are given below) necessary to direct expression of the coding sequences; (c) expression vectors encoding, in addition to a Arp-Tl polypeptide, a sequence unrelated to Arp-Tl, such as a reporter, a marker, or a signal peptide fused to Arp- Tl; and (d) genetically engineered host cells (see below) that contain any of the foregoing expression vectors and thereby express the nucleic acid molecules of the invention.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • a DNA or RNA sequence e.g. a foreign gene
  • Any expression vector for animal cell can be used.
  • suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al, 1984), pKCR (O'Hare et al, 1981), pSGl beta d2-4 (Miyaji et al, 1990) and the like.
  • Plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vectors include adenoviral, retroviral, herpes virus and AAV vectors.
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • WO 95/14785 WO 96/22378
  • US 5,882,877 US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
  • the transcriptional/translational regulatory elements referred to above and further described below include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression.
  • Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast a-mating factors.
  • Recombinant nucleic acid molecules can contain a sequence encoding Arp-Tl or Arp- Tl having an heterologous signal sequence.
  • the full length Arp-Tl polypeptide, or a fragment thereof, may be fused to such heterologous signal sequences or to additional polypeptides, as described below.
  • the nucleic acid molecules of the invention can encode the mature form of Arp-Tl or a form that includes an exogenous polypeptide that facilitates secretion.
  • the nucleic acid can form part of a hybrid gene encoding additional polypeptide sequences, for example, a sequence that functions as a marker or reporter.
  • marker and reporter genes include ⁇ -lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydro folate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding ⁇ -galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).
  • CAT chloramphenicol acetyltransferase
  • ADA adenosine deaminase
  • DHFR dihydro folate reductase
  • HPH hygromycin-B-phosphotransferase
  • TK thymidine kinase
  • lacZ encoding ⁇ -galactosidas
  • the hybrid polypeptide will include a first portion and a second portion; the first portion being a Arp-Tl polypeptide and the second portion being, for example, the reporter described above or an Ig constant region or part of an Ig constant region, e.g., the CH2 and CH3 domains of IgG2a heavy chain.
  • Other hybrids could include an antigenic tag or His tag to facilitate purification.
  • the expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (for example, E. coli and B. subtilis) to transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention; yeast (for example, Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the nucleic acid molecule of the invention; insect cell systems infected with recombinant virus expression vectors (for example, baculo virus) containing the nucleic acid molecule of the invention; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid) containing a ACTRT1 nucleotide sequence; or mammalian cell systems (for example, COS, CHO, B
  • the methods as described above can be performed in vitro, in vivo, or ex vivo.
  • In vitro application of Arp-Tl can be useful, for example, in basic scientific studies of tumor cell biology, e.g., studies on signal transduction or cell cycle analysis.
  • the methods of the invention will preferably be performed in vivo for the treatment of cancer.
  • a further aspect of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a polypeptide as described above, a nucleic acid molecule as described above or a vector as described above.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the Arp-Tl polypeptide (or a variant or a functional fragment thereof) itself is administered to the subject.
  • the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. They are preferably delivered directly to tumor cells, e.g., to a tumor or a tumor bed following surgical excision of the tumor, in order to kill any remaining tumor cells.
  • a pharmaceutically-acceptable carrier e.g., physiological saline
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 ⁇ g/kg. Wide variations in the needed dosage are to be expected in view of the variety of polypeptides and fragments available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
  • Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
  • Encapsulation of the polypeptide in a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
  • a polynucleotide containing a nucleic acid sequence encoding a Arp-Tl polypeptide, a variant thereof or a functional fragment thereof can be delivered to cancer cells in a mammal.
  • Expression of the coding sequence will preferably be directed to lymphoid tissue of the subject by, for example, delivery of the polynucleotide to the lymphoid tissue.
  • Expression of the coding sequence can be directed to any cell in the body of the subject. However, expression will preferably be directed to cells in the vicinity of the tumor cells whose proliferation it is desired to inhibit.
  • expression of the coding sequence can be directed to the tumor cells themselves. This can be achieved by, for example, the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.
  • liposomes prepared by standard methods.
  • the vectors can be incorporated alone into these delivery vehicles or co- incorporated with tissue-specific or tumor- specific antibodies.
  • tissue-specific or tumor- specific antibodies Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells.
  • tissue specific targeting can be achieved by the use of tissue- specific transcriptional regulatory elements (TRE) which are known in the art.
  • Delivery of "naked DNA" i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
  • the nucleic acid sequence encoding the Arp-Tl polypeptide or functional fragment of interest with an initiator methionine and optionally a targeting sequence is operatively linked to a promoter or enhancer-promoter combination.
  • Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription initiation site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site. To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3' ) of the promoter. The coding sequence of the expression vector is operatively linked to a transcription terminating region.
  • Suitable expression vectors include plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.
  • said vectors are as described above.
  • Polynucleotides can be administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles that are suitable for administration to a human, e.g., physiological saline or liposomes.
  • a therapeutically effective amount is an amount of the polynucleotide that is capable of producing a medically desirable result (e.g., decreased proliferation of cancer cells) in a treated animal.
  • the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a preferred dosage for administration of polynucleotide is from approximately 106 to 1012 copies of the polynucleotide molecule. This dose can be repeatedly administered, as needed. Routes of administration can be any of those listed above. Screening methods:
  • the present invention also relates to a method for screening a plurality of candidate compounds useful for treating cancer comprising the steps consisting of (a) testing each of the candidate compounds for its ability to enhancing ACTRTl gene expression and (b) and positively selecting the candidate compounds capable of enhancing said expression.
  • Testing whether a candidate compound can enhance ACTRTl gene expression can be determined using or routinely modifying reporter assays known in the art.
  • the method may involve contacting cells expressing ACTRTl with the candidate compound, and measuring the ACTRTl mediated transcription (e.g., activation of promoters containing ACTRTl binding sites), and comparing the cellular response to a standard cellular response.
  • the standard cellular response is measured in absence of the candidate compound.
  • the determination comprises contacting the cells with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth.
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate may be a solid or semi- so lid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • the expression level may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • the screening method of the present invention involves transfecting a eukaryotic cell with DNA wherein a reporter gene (.e.g, GFP) is linked to the promoter region of ACTRTl, in which case, the amount of transcription from the reporter gene may be measured by assaying the level of reporter gene product, or the level of activity of the reporter gene product in the case where the reporter gene is an enzyme. An increase in the amount the expression of the reporter gene would indicate that the candidate compound is capable of enhancing ACTRTl gene expression.
  • a reporter gene .e.g, GFP
  • the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides.
  • Other potential candidate compounds include antisense molecules.
  • the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in inhibiting the proliferation and migration of cancer cells.
  • the screening method may further comprise the steps of i) bringing into contact a cancer cell with a positively selected candidate compound ii) determining proliferation and/or migration level of said cancer cell and iii) comparing the level determined at step ii) with the level determined when step i) is performed in the absence of the positively selected candidate compound and iv) positively selecting the candidate compound when the level determined at step i) is lower than the level determined in the absence of the candidate compound.
  • Step i) as above described may be performed by adding an amount of the candidate compound to be tested to the culture medium of the cancer cells.
  • a plurality of culture samples are prepared, so as to add increasing amounts of the candidate compound to be tested in distinct culture samples.
  • at least one culture sample without candidate compound is also prepared as a control for further comparison.
  • the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on cancer animal models as described in the EXAMPLE.
  • the invention also features diagnostic assays. Such assays are based on the basis that: the hACTRTl gene is either not expressed or is poorly expressed in cancer while it is highly expressed in normal tissue (non tumor tissue) (i.e. ACTRTl deficiency). Thus, findings of no or low expression of the ACTRTl gene in test cells would indicate that the test cells are cancer cells. Such tests can be used on their own or, preferably, in conjunction with other procedures to test for cancer in appropriate subjects.
  • the present invention thus relates to a method for determining whether a subject has a or is at risk of having cancer comprising the steps consisting of detecting an ACTRTl deficiency in the a biological sample obtained from the subject and concluding that the subject has a cancer or is at risk of having a cancer when an ACTRTl deficiency is detected.
  • ACTRTl deficiency denotes that the cancer cells of the subject or a part thereof have an ACTRTl dysfunction, a low or a null expression of ACTRTl .
  • Said deficiency may typically result from a mutation in the ACTRTl so that the pre-ARNm is degraded through the NMD (non sense mediated decay) system.
  • Said deficiency may also typically result from a mutation so that the protein is mis folded and degraded through the proteasome. Said deficiency may also result from a loss of function mutation leading to a dysfunction of the protein. Said deficiency may also result from an epigenetic control of gene expression (e.g. methylation) so that the gene is less expressed in the cells of the subject. Said deficiency may also result from a repression of the ACTRTl gene induce by a particular signalling pathway. Said deficiency may also result from a mutation in a nucleotide sequence that control the expression of ACTRTl . Typically said sequence may be a conserved non coding element, such as CNE12 as described in EXAMPLE.
  • the biological sample is a blood sample or a PBMC sample or is a tissue sample resulting from a biopsy (e.g. biopsy performed in the tissue suspected to be cancerous)
  • the first step comprises measuring the number of methylated C residues in the CpG sequences within the CpG island of the ACTRTl promoter.
  • Methods of measuring the number of methylated C residues in the CpG sequences within the CpG island of a gene promoter are known in the art. Standardizing such methylation assays to discriminate between cancer and non-cancer cells of interest would involve experimentation familiar to those in the art. For example, the methylation status of the ACTRTl promoter region in DNA from sample cancer cells of interest obtained from a large number of patients can be compared to the methylation status of the ACTRTl promoter region in DNA from normal cells corresponding to the cancer cells obtained either from the same patients or from normal individuals.
  • methylation status of the ACTRTl promoter region in DNA from cancer cells of each patient can be compared to the methylation status of the ACTRTl promoter region in DNA from normal cells (corresponding to the cancer cells) obtained from the same patient.
  • methylation of as few as one cytosine residue could discriminate between cancer and non-cancer cells.
  • Other methods for quantitating methylation of DNA are known in the art.
  • Such methods are based on: (a) the inability of methylation-sensitive restriction enzymes to cleave sequences that contain one or more methylated CpG sites [Issa et al. (1994) Nat. Genet. 7:536-540; Singer-Sam et al. (1990) Mol. Cell. Biol. 10:4987-4989; Razin et al. (1991) Microbiol. Rev. 55:451-458; Stoger et al. (1993) Cell 73:61-71]; and (b) the ability of bisulfite to convert cytosine to uracil and the lack of this ability of bisulfite on methylated cytosine [Frommer et al. (1992) Proc. Natl.
  • the first step consists in detecting the mutation that is responsible for the ACTRT1 deficiency.
  • nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance.
  • the nucleic acid sample may be obtained from any cell source or tissue biopsy. Non-limiting examples of cell sources available include without limitation blood cells, buccal cells, epithelial cells, fibroblasts, or any cells present in a tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, such as blood, plasma, serum, lymph, etc.
  • DNA may be extracted using any methods known in the art, such as described in Sambrook et al, 1989.
  • RNA may also be isolated, for instance from tissue biopsy, using standard methods well known to the one skilled in the art such as guanidium thiocyanate-phenol-chloroform extraction. Mutations may be detected in a RNA or DNA sample, preferably after amplification.
  • the isolated RNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a mutated site or that enable amplification of a region containing the mutated site.
  • reverse transcription and amplification such as reverse transcription and amplification by polymerase chain reaction (RT-PCR)
  • RT-PCR polymerase chain reaction
  • conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a particular mutation.
  • RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • a cDNA obtained from RNA may be cloned and sequenced to identify a mutation in ACTRT1 sequence.
  • numerous strategies for genotype analysis are available (Antonarakis et al, 1989 ; Cooper et al., 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site.
  • a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation.
  • Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997).
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific oligonucleotides
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • InvaderTMassay or oligonucleotide ligation assay (OLA).
  • OLA may be used for revealing base substitution mutations.
  • oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation.
  • DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions where the mutations are located.
  • Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.
  • the mutation may be also detected at a protein level (e.g.
  • a biological sample such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of a mutated form of ACTRT1 protein, i.e. antibodies that are capable of distinguishing between a mutated form of ACTRT1 and the wild-type protein, to determine the presence or absence of a ACTRT1 specified by the antibody.
  • the antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v).
  • the first step consists in determining the expression level of
  • the first step consist in i) determining the expression level of ACTRT1 gene, ii) comparing the level determined at i) with a predetermined reference value and iii) concluding that the subject has a ACTRT1 deficiency when the expression level determined at i) is lower than the predetermined reference value.
  • the predetermined reference value is the expression level determined in a healthy population of subject (e.g. the mean expression).
  • the expression level of a gene may be determined by determining the quantity of mRNA.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a "detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315- 22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • SISH silver in situ hybridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox- inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC-conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podo
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).
  • the nCounter® Analysis system is used to detect intrinsic gene expression.
  • the basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3): 317-325; the contents of which are each incorporated herein by reference in their entireties).
  • the code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed.
  • a pair of probes is designed for each DNA or RNA target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode.
  • the reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over- lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence.
  • each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively.
  • the capture probe can comprise a second target-specific sequence; and a first affinity tag.
  • the capture probe can also comprise one or more label attachment regions.
  • the first target- specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected. Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library".
  • the relative abundance of each target is measured in a single multiplexed hybridization reaction.
  • the method comprises contacting the tumor sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex.
  • the complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution.
  • the tripartite hybridized complexes are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes. This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample.
  • All post hybridization steps are handled robotically on a custom liquid-handling robot (Prep Station, NanoString Technologies).
  • Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe,electrophoresed to elongate the reporter probes, and immobilized.
  • the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies).
  • the expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface.
  • FOV fields-of-view
  • Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance. Data is output in simple spreadsheet format listing the number of counts per target, per sample.
  • This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No. WO 07/076129 and WO07/076132, and US Patent Publication No. 2010/0015607 and 2010/0261026, the contents of which are incorporated herein in their entireties. Further, the term nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No.2010/0047924, incorporated herein by reference in its entirety.
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • Other methods for determining the expression level of a gene include the determination of the quantity of proteins encoded by said genes.
  • Such methods comprise contacting the sample with a binding partner capable of selectively interacting with a marker protein present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the binding partner may also be an aptamer.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • IHC immunohistochemistry
  • IHC specifically provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest.
  • a sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy.
  • Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling.
  • IHC systems include, for example, EnVision(TM) (DakoCytomation), Powervision(R) (Immunovision, Springdale, AZ), the NBA(TM) kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine(R) (Nichirei Corp, Tokyo, Japan).
  • a tissue section (e.g. a sample comprising cumulus cells) may be mounted on a slide or other support after incubation with antibodies directed against the proteins encoded by the genes of interest. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the proteins of interest.
  • IHC samples may include, for instance: (a) preparations comprising cumulus cells (b) fixed and embedded said cells and (c) detecting the proteins of interest in said cells samples.
  • an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.
  • BCC Cutaneous basal cell carcinoma
  • BCCs Cutaneous basal cell carcinoma
  • Most BCCs are sporadic but some forms are inherited.
  • Bazex-Dupre-Christol syndrome (BDCS) is an X-linked dominant predisposition to BCCs with typical skin manifestations, suggestive of a primary hair follicle anomaly 1 ' 2 ' 3 ' 4 .
  • ACTRTI expression in HEK293T cells showed that the mutant cDNA encodes a 25 kDa truncated protein. Yet, no mutation of the coding region of ACTRTI was found in the four other families linked to the same interval. No additional exons in ACTRTI were found by 5 '- 3' RACE-PCR and no rearrangements in the candidate region were identified in the remaining 4/6 patients by high-density tiling-path comparative genomic hybridization array.
  • CNEs non-coding elements 6
  • CNE 12 This unusually long CNE (2.5 kb) is conserved in mammals (61% identity with the mouse sequence) and teleost fish. In silico predicted secondary structure of CNE12 suggested that it may be transcribed 6 ' 11 .
  • RT-PCR and in situ hybridization (ISH) experiments showed that CNE 12 was indeed expressed in epidermis and epidermal appendages but not in the dermis.
  • ISH of skin sections showed complete abolition of the specific ncRNA in the two male patients carrying the CNE 12 variation, demonstrating the drastic impact of the g.l27372937A>T variation on its stability or expression.
  • ISH analysis detected CNE 12 non-coding RNAs (ncRNAs) in a patient carrying the c.547-548 InsA ACTRTI mutation.
  • ncRNAs non-coding RNAs
  • eRNAs enhancers
  • IHC Immuno-histochemical
  • Arp-Tl belongs to the Actin-Related Proteins (Arps) family. Nuclear Arps are essential elements of the macromolecular machinery that controls chromatin remodeling, dynamic changes in DNA structure, transcription and re air 15 ' 16 ' 17 ' 18 ' 19 ' 20 ' 21 - 22 . Ultra-thin sections of normal skin processed for transmission electronic microscopy analyses detected Arp-Tl in both nucleus and cytoplasm. Subcellular protein fractionation assays showed that Arp-Tl binds chromatin, while the truncated Arp-Tl protein was absent in the chromatin- bound fraction. Arp-Tl chromatin-binding in controls but not BDCS is consistent with its putative involvement in chromatin remodeling and its alteration in the disease.
  • HEK293T cells were transiently transfected with either wild type or mutant c.547_548 InsA ACTRTI constructs.
  • the wild type but not the mutant construct significantly decreased cell proliferation, by arresting cells in S-phase.
  • Germline mutations in the ACTRTI gene and its non-coding surrounding elements in BCCs represent a hitherto unreported mechanism of inherited predisposition to skin tumors. This study highlights the impact of intergenic mutations in human diseases. Elucidating the disease mechanism in BDCS, a rare inherited condition, has thus shed light on a most common human cancer, BCC. Developing novel Hedgehog signaling inhibitors targeting Arp- Tl will hopefully help improving treatment of this frequent, potentially devastating condition.

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Abstract

The present invention relates to a novel tumor suppressor gene and uses thereof for the treatment and diagnosis of cancer. In particular, the present invention relates to a method which involves contacting a cancer cell with an Arp-T1 polypeptide of the invention, or a functional variant or fragment thereof, in order to inhibit proliferation and migration of the cancer cell.

Description

NOVEL TUMOR SUPPRESSOR GENE AND USES THEREOF FOR THE
TREATMENT AND DIAGNOSIS OF CANCER
FIELD OF THE INVENTION:
The present invention relates to a novel tumor suppressor gene and uses thereof for the treatment and diagnosis of cancer.
BACKGROUND OF THE INVENTION:
A tumor suppressor gene is a gene that protects a cell from one step on the path to cancer. When this gene mutates to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes. The loss of these genes may be even more important than proto-oncogene/oncogene activation for the formation of many kinds of human cancer cells. For instance, tumor- suppressor genes may have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. Examples of tumor suppressor genes include but are not limited to TP53, PTEN, pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14.
SUMMARY OF THE INVENTION:
The present invention relates to a novel tumor suppressor gene and uses thereof for the treatment of cancer. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Cutaneous basal cell carcinoma (BCCs) is the most common human cancer. While most cases are sporadic, some forms are inherited, as in Bazex-Dupre-Christol syndrome (BDCS), an X-linked dominant cancer-prone genodermatosis. Studying a series of 6 BDCS families, the inventors found a mutation in a gene encoding the actin-related protein Arp-Tl (ACTRTl) in 2/6 BDCS patients, resulted in absent expression of Arp-Tl in BDCS tumors. Wild-type Arp-Tl, but not its mutant counterpart could bind chromatin. Overexpression of ACTRTl resulted in an increased number of cells arrested in S-phase, which supports a role for Arp-Tl in cell cycle regulation. When stably expressed in MDA-MB231 cell line, ACTRTl reduced proliferation and migration. Moreover, xenografts of ACTRTl -MDA- MB231 cells in nude mice confirmed the ability of ACTRTl to inhibit tumor growth. Arp-Tl was found to act as a tumor-suppressor via its direct binding to Hedgehog signaling target genes, thus inhibiting their expression.
As used herein, the term "ACTRT1" has its general meaning in the art and refers to the Actin-Related Protein Tl . An exemplary amino acid sequence is SEQ ID NO: l and an exemplary nucleic acid sequence is SEQ ID NO:2.
SEQ ID NO: l (NCBI Reference Sequence: NP 612146.1)
mfhphaldvp avifdngsgl ckaglsgeig prhvissvlg hckfnvplar lnqkyfvgqe alykyealhl hypierglvt gwddmeklwk hlferelgvk psqqpvlmte pslnpreire klaemmfetf svpgfylsnh avaalyasac vtglwdsgd gvtctvpife gyslphavtk lcmagrdite hltrllfasg fnfpcilnka wnnikeklc yialepekel rksrgevlga yrlpdghvih fgdelyqvpe vlfapdqlgi hspglskmvs ssimkcdtdi qnklyadivl sggttllpgl eerlmkeveq laskgtpiki taspdrcfsa wigasimtsm ssfkqmwvts adfkeygtsv vqrrcf
SEQ ID NO:2 (NCBI Reference Sequence: NM 138289.3):
agaatggaga taagattcag aggttgagga tggggtgtcc tggtggactg aaggtagcct actagctgta catgggtgac aacttgaaac ttcagaaccc tgaagtttaa aaaattctaa aggtgcctgt catctcagag agtgacgtaa gtgttctttc tttatttggg ggaagtccag gagaacatat tacagacatg tttaatccac atgcattaga tgttcctgct gtaatttttg acaatggttc aggactctgc aaagcaggcc tgtctggaga gattggaccc cgccatgtca tcagctccgt cttgggacat tgtaaattca atgtgccttt agcaagactt aatcagaagt acttcgtggg gcaagaagcc ctgtacaagt atgaggccct acatttgcac taccccattg agcgtggact ggtaacagga tgggatgaca tggagaaact ctggaaacat ctctttgagc gggagcttgg agtaaaaccc agccaacagc ctgtacttat gaccgagccc tctttgaatc ctagggaaat tcgagaaaag ctagcagaaa tgatgtttga gaccttcagt gtgcctggtt tctacctgtc taatcatgcg gtggcagcgc tctatgcctc tgcctgtgtc acaggcctgg tggtggacag tggagatggg gtcacttgca ctgtccccat ctttgagggt tactccctgc ctcacgcagt caccaaactc tgtatggcag ggagggacat cacagagcac ctcacccggc tcctctttgc tagcgggttt aacttccctt gcatactcaa caaggccgtg gtaaataaca tcaaagagaa gttgtgctac atcgccttgg agccagagaa agagctacgc aagagccggg gagaggtcct gggagcatac agactgccag atggacatgt catccacttt ggggatgagc tgtaccaagt gcccgaggtt ctttttgcac ctgaccagct gggcatccac agcccaggac tctcaaaaat ggtctccagc agcatcatga agtgtgacac tgacatccag aataaacttt atgcagacat tgtactctcc gggggcacca ctctcctccc tgggctggag gaaaggctca tgaaggaagt ggaacagctg gcttccaaag gtactcccat caagatcaca gcttctcctg atagatgctt ctctgcatgg attggtgcat ccatcatgac ctctatgagc agtttcaagc agatgtgggt cacctcggca gacttcaagg agtatgggac atctgtggtt caaagaaggt gcttttaaag atccttgagc agaggagaca tcttgaagtg tcagattaca ggagtaccag tgggagatgg cattttcttc tgggcttcag catgatgttc aataaaagtt ttgccatttc aaaaaaaaaa aa Methods of treatment:
A first object of the present invention relates to a method which involves contacting a cancer cell with an Arp-Tl polypeptide of the invention, or a functional variant or fragment thereof, in order to inhibit proliferation and migration of the cancer cell.
The polypeptides of the invention include all those disclosed herein. They can be, for example, bArp-Tl (SEQ ID NO: l) and functional variant or fragments of this polypeptide. The polypeptides embraced by the invention also include fusion proteins that contain either full-length Arp-Tl or a functional fragment of it fused to unrelated amino acid sequence. The unrelated sequences can be additional functional domains or signal peptides. Signal peptides are described in greater detail and exemplified below. The polypeptides can be any of those described above but with one or more (e.g., 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11 ; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21 ; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71 ; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; or more) conservative substitutions.
In some embodiments, the functional variant consists of a polypeptide having a sequence having at least 70% of identity with SEQ ID NO: l . According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91 ; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the polypeptide of the invention is a functional conservative variant of the polypeptide. As used herein the term "function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared (i.e. inhibition of migration and proliferation of cancer cells). Functional properties of the polypeptide of the invention could typically be assessed in any functional assay as described in the EXAMPLE.
In some embodiments, the fragment comprises at least 10; 11; 12; 13; 14; 14; 15; 16;
17; 18; 19; 20; 21 ; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64;
65; 66; 67; 68; 69; 70; 71 ; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88;
89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109;
110; 111; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127;
128; 129; 130; 131; 132; 133; 134; 135; 136; 137; 138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161; 162; 163;
164; 165; 166; 167; 168; 169; 170; 171; 172; 173; 174; 175; 176; 177; 178; 179; 180; 181 ;
182; 183; 184; 185; 186; 187; 188; 189; 190; 191; 192; 193; 194; 195; 196; 197; 198; 199;
200; 201; 202; 203; 204; 205; 206; 207; 208; 209; 210; 211; 212; 213; 214; 215; 216; 217;
218; 219; 220; 221; 222; 223; 224; 225; 226; 227; 228; 229; 230; 231; 232; 233; 234; 235; 236; 237; 238; 239; 240; 241; 242; 243; 244; 245; 246; 247; 248; 249; 250; 251; 252; 253;
254; 255; 256; 257; 258; 259; 260; 261; 262; 263; 264; 265; 266; 267; 268; 269; 270; 271 ;
272; 273; 274; 275; 276; 277; 278; 279; 280; 281; 282; 283; 284; 285; 286; 287; 288; 289;
290; 291; 292; 293; 294; 295; 296; 297; 298; 299; 300; 301; 302; 303; 304; 305; 306; 307;
308; 309; 310; 311; 312; 313; 314; 315; 316; 317; 318; 319; 320; 321; 322; 323; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 342; 343;
344; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 357; 358; 359; 360; 361 ;
362; 363; 364; 365; 366; 367; 368; 369; 370; 371; 372; 373; 374 or 375 consecutive amino acids of SEQ ID NO: l .
The polypeptides can be purified from natural sources (e.g., any cell that naturally produces Arp-Tl polypeptides). Smaller peptides (e.g. less than 50 amino acids long) can also be conveniently synthesized by standard chemical means. In addition, both polypeptides and peptides can be produced by standard in vitro recombinant DNA techniques and in vivo transgenesis, using nucleotide sequences encoding the appropriate polypeptides or peptides. Methods well-known to those skilled in the art can be used to construct expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals. Polypeptides and fragments of the invention also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
Also of interest are peptidomimetic compounds that are designed based upon the amino acid sequences of the functional peptide fragments. Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide. The peptide motif provides the peptidomimetic compound with the ability to inhibit the proliferation of cancer cells in a manner qualitatively identical to that of the Arp-Tl functional fragment from which the peptidomimetic was derived. Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased cell permeability and prolonged biological half-life. The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics. A further aspect of the present invention relates to a method which involves contacting a cancer cell with an ACTRT1 nucleic acid molecule of the invention, in order to inhibit proliferation and migration of the cancer cell. According to the invention an "ACTRT1 nucleic acid molecule" is a nucleic acid molecule which encodes a hArp-Tl (e.g. SEQ ID NO: l) polypeptide as well as functional variants or fragments of this polypeptide as described above.
The ACTRT1 nucleic acid molecules of the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Segments of these molecules are also considered within the scope of the invention, and can be produced by, for example, the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases. A ribonucleic acid (RNA) molecule can be produced by in vitro transcription. The nucleic acid molecules of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide (for example, the polypeptides with SEQ ID NO: l). In addition, these nucleic acid molecules are not limited to coding sequences, e.g., they can include some or all of the non-coding sequences that lie upstream or downstream from a coding sequence.
The nucleic acid molecules of the invention can be synthesized (for example, by phosphoramidite-based synthesis) or obtained from a biological cell, such as the cell of a mammal. The nucleic acids can be those of a human, non-human primate (e.g., monkey), mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat. Combinations or modifications of the nucleotides within these types of nucleic acids are also encompassed.
In addition, the isolated nucleic acid molecules of the invention encompass segments that are not found as such in the natural state. Thus, the invention encompasses recombinant nucleic acid molecules (for example, isolated nucleic acid molecules encoding hArp-Tl) incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location). In some embodiments, the nucleic acid molecule of the invention is SEQ ID NO:2 or a nucleic acid sequence having at least 70% of identity with SEQ ID NO:2.
The invention also encompasses: (a) vectors that contain any of the foregoing ACTRT1 nucleic acid molecules; (b) expression vectors that contain any of the foregoing ACTRT1 nucleic acid molecules operably linked to any transcriptional/translational regulatory elements (examples of which are given below) necessary to direct expression of the coding sequences; (c) expression vectors encoding, in addition to a Arp-Tl polypeptide, a sequence unrelated to Arp-Tl, such as a reporter, a marker, or a signal peptide fused to Arp- Tl; and (d) genetically engineered host cells (see below) that contain any of the foregoing expression vectors and thereby express the nucleic acid molecules of the invention.
As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al, 1984), pKCR (O'Hare et al, 1981), pSGl beta d2-4 (Miyaji et al, 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478. The transcriptional/translational regulatory elements referred to above and further described below include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast a-mating factors.
Recombinant nucleic acid molecules can contain a sequence encoding Arp-Tl or Arp- Tl having an heterologous signal sequence. The full length Arp-Tl polypeptide, or a fragment thereof, may be fused to such heterologous signal sequences or to additional polypeptides, as described below. Similarly, the nucleic acid molecules of the invention can encode the mature form of Arp-Tl or a form that includes an exogenous polypeptide that facilitates secretion. Similarly, the nucleic acid can form part of a hybrid gene encoding additional polypeptide sequences, for example, a sequence that functions as a marker or reporter. Examples of marker and reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydro folate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter. Generally, the hybrid polypeptide will include a first portion and a second portion; the first portion being a Arp-Tl polypeptide and the second portion being, for example, the reporter described above or an Ig constant region or part of an Ig constant region, e.g., the CH2 and CH3 domains of IgG2a heavy chain. Other hybrids could include an antigenic tag or His tag to facilitate purification.
The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (for example, E. coli and B. subtilis) to transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention; yeast (for example, Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the nucleic acid molecule of the invention; insect cell systems infected with recombinant virus expression vectors (for example, baculo virus) containing the nucleic acid molecule of the invention; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid) containing a ACTRT1 nucleotide sequence; or mammalian cell systems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter and the vaccinia virus 7.5K promoter). Also useful as host cells are primary or secondary cells obtained directly from a mammal and transfected with a plasmid vector or infected with a viral vector.
The methods as described above can be performed in vitro, in vivo, or ex vivo. In vitro application of Arp-Tl can be useful, for example, in basic scientific studies of tumor cell biology, e.g., studies on signal transduction or cell cycle analysis. However, the methods of the invention will preferably be performed in vivo for the treatment of cancer.
Accordingly a further aspect of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a polypeptide as described above, a nucleic acid molecule as described above or a vector as described above.
As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; muco epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In one in vivo approach, the Arp-Tl polypeptide (or a variant or a functional fragment thereof) itself is administered to the subject. Generally, the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. They are preferably delivered directly to tumor cells, e.g., to a tumor or a tumor bed following surgical excision of the tumor, in order to kill any remaining tumor cells. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Wide variations in the needed dosage are to be expected in view of the variety of polypeptides and fragments available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the polypeptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
Alternatively, a polynucleotide containing a nucleic acid sequence encoding a Arp-Tl polypeptide, a variant thereof or a functional fragment thereof can be delivered to cancer cells in a mammal. Expression of the coding sequence will preferably be directed to lymphoid tissue of the subject by, for example, delivery of the polynucleotide to the lymphoid tissue. Expression of the coding sequence can be directed to any cell in the body of the subject. However, expression will preferably be directed to cells in the vicinity of the tumor cells whose proliferation it is desired to inhibit. In certain embodiments, expression of the coding sequence can be directed to the tumor cells themselves. This can be achieved by, for example, the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.
Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The vectors can be incorporated alone into these delivery vehicles or co- incorporated with tissue-specific or tumor- specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. Alternatively, tissue specific targeting can be achieved by the use of tissue- specific transcriptional regulatory elements (TRE) which are known in the art. Delivery of "naked DNA" (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
In the relevant polynucleotides (e.g., expression vectors), the nucleic acid sequence encoding the Arp-Tl polypeptide or functional fragment of interest with an initiator methionine and optionally a targeting sequence is operatively linked to a promoter or enhancer-promoter combination.
Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription initiation site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site. To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3' ) of the promoter. The coding sequence of the expression vector is operatively linked to a transcription terminating region.
Suitable expression vectors include plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others. Typically, said vectors are as described above.
Polynucleotides can be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles that are suitable for administration to a human, e.g., physiological saline or liposomes. A therapeutically effective amount is an amount of the polynucleotide that is capable of producing a medically desirable result (e.g., decreased proliferation of cancer cells) in a treated animal. As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for administration of polynucleotide is from approximately 106 to 1012 copies of the polynucleotide molecule. This dose can be repeatedly administered, as needed. Routes of administration can be any of those listed above. Screening methods:
The present invention also relates to a method for screening a plurality of candidate compounds useful for treating cancer comprising the steps consisting of (a) testing each of the candidate compounds for its ability to enhancing ACTRTl gene expression and (b) and positively selecting the candidate compounds capable of enhancing said expression.
Testing whether a candidate compound can enhance ACTRTl gene expression can be determined using or routinely modifying reporter assays known in the art. For example, the method may involve contacting cells expressing ACTRTl with the candidate compound, and measuring the ACTRTl mediated transcription (e.g., activation of promoters containing ACTRTl binding sites), and comparing the cellular response to a standard cellular response. Typically, the standard cellular response is measured in absence of the candidate compound. An increased cellular response over the standard indicates that the candidate compound is able to enhance ACTRTl gene expression. Determination of the expression level of a gene can be performed by a variety of techniques. Typically, the determination comprises contacting the cells with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi- so lid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample. In a preferred embodiment, the expression level may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
In some embodiments, the screening method of the present invention involves transfecting a eukaryotic cell with DNA wherein a reporter gene (.e.g, GFP) is linked to the promoter region of ACTRTl, in which case, the amount of transcription from the reporter gene may be measured by assaying the level of reporter gene product, or the level of activity of the reporter gene product in the case where the reporter gene is an enzyme. An increase in the amount the expression of the reporter gene would indicate that the candidate compound is capable of enhancing ACTRTl gene expression.
In a particular embodiment, the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides. Other potential candidate compounds include antisense molecules.
The candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in inhibiting the proliferation and migration of cancer cells. Typically, the screening method may further comprise the steps of i) bringing into contact a cancer cell with a positively selected candidate compound ii) determining proliferation and/or migration level of said cancer cell and iii) comparing the level determined at step ii) with the level determined when step i) is performed in the absence of the positively selected candidate compound and iv) positively selecting the candidate compound when the level determined at step i) is lower than the level determined in the absence of the candidate compound. Step i) as above described may be performed by adding an amount of the candidate compound to be tested to the culture medium of the cancer cells. Usually, a plurality of culture samples are prepared, so as to add increasing amounts of the candidate compound to be tested in distinct culture samples. Generally, at least one culture sample without candidate compound is also prepared as a control for further comparison. Finally, the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on cancer animal models as described in the EXAMPLE.
Methods for diagnosis:
The invention also features diagnostic assays. Such assays are based on the basis that: the hACTRTl gene is either not expressed or is poorly expressed in cancer while it is highly expressed in normal tissue (non tumor tissue) (i.e. ACTRTl deficiency). Thus, findings of no or low expression of the ACTRTl gene in test cells would indicate that the test cells are cancer cells. Such tests can be used on their own or, preferably, in conjunction with other procedures to test for cancer in appropriate subjects. The present invention thus relates to a method for determining whether a subject has a or is at risk of having cancer comprising the steps consisting of detecting an ACTRTl deficiency in the a biological sample obtained from the subject and concluding that the subject has a cancer or is at risk of having a cancer when an ACTRTl deficiency is detected. In the context of the invention, the term "ACTRTl deficiency" denotes that the cancer cells of the subject or a part thereof have an ACTRTl dysfunction, a low or a null expression of ACTRTl . Said deficiency may typically result from a mutation in the ACTRTl so that the pre-ARNm is degraded through the NMD (non sense mediated decay) system. Said deficiency may also typically result from a mutation so that the protein is mis folded and degraded through the proteasome. Said deficiency may also result from a loss of function mutation leading to a dysfunction of the protein. Said deficiency may also result from an epigenetic control of gene expression (e.g. methylation) so that the gene is less expressed in the cells of the subject. Said deficiency may also result from a repression of the ACTRTl gene induce by a particular signalling pathway. Said deficiency may also result from a mutation in a nucleotide sequence that control the expression of ACTRTl . Typically said sequence may be a conserved non coding element, such as CNE12 as described in EXAMPLE. Typically, the biological sample is a blood sample or a PBMC sample or is a tissue sample resulting from a biopsy (e.g. biopsy performed in the tissue suspected to be cancerous)
In some embodiments, the first step comprises measuring the number of methylated C residues in the CpG sequences within the CpG island of the ACTRTl promoter. Methods of measuring the number of methylated C residues in the CpG sequences within the CpG island of a gene promoter are known in the art. Standardizing such methylation assays to discriminate between cancer and non-cancer cells of interest would involve experimentation familiar to those in the art. For example, the methylation status of the ACTRTl promoter region in DNA from sample cancer cells of interest obtained from a large number of patients can be compared to the methylation status of the ACTRTl promoter region in DNA from normal cells corresponding to the cancer cells obtained either from the same patients or from normal individuals. From such experimentation it will be possible to establish a range of "cancer levels" of methylation and a range of "normal levels" of methylation. Alternatively, the methylation status of the ACTRTl promoter region in DNA from cancer cells of each patient can be compared to the methylation status of the ACTRTl promoter region in DNA from normal cells (corresponding to the cancer cells) obtained from the same patient. In such assays, it is possible that methylation of as few as one cytosine residue could discriminate between cancer and non-cancer cells. Other methods for quantitating methylation of DNA are known in the art. Such methods are based on: (a) the inability of methylation-sensitive restriction enzymes to cleave sequences that contain one or more methylated CpG sites [Issa et al. (1994) Nat. Genet. 7:536-540; Singer-Sam et al. (1990) Mol. Cell. Biol. 10:4987-4989; Razin et al. (1991) Microbiol. Rev. 55:451-458; Stoger et al. (1993) Cell 73:61-71]; and (b) the ability of bisulfite to convert cytosine to uracil and the lack of this ability of bisulfite on methylated cytosine [Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-1831; Myohanen et al. (1994) DNA Sequence 5: 1-8; Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826; Gonzalgo et al. (1997) Nucleic Acids Res. 25:2529-2531; Sadri et al. (1996) Nucleic Acids Res. 24:5058-5059; Xiong et al. (1997) Nucleic Acids Res. 25:2532- 2534]. In some embodiments, the first step consists in detecting the mutation that is responsible for the ACTRT1 deficiency. One skilled in the art can easily identify a mutation in ACTRT1 gene or in a conserved non coding element, such as CNE12. Typically the mutation may be detected by analyzing nucleic acid molecule. In the context of the invention, nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance. The nucleic acid sample may be obtained from any cell source or tissue biopsy. Non-limiting examples of cell sources available include without limitation blood cells, buccal cells, epithelial cells, fibroblasts, or any cells present in a tissue obtained by biopsy. Cells may also be obtained from body fluids, such as blood, plasma, serum, lymph, etc. DNA may be extracted using any methods known in the art, such as described in Sambrook et al, 1989. RNA may also be isolated, for instance from tissue biopsy, using standard methods well known to the one skilled in the art such as guanidium thiocyanate-phenol-chloroform extraction. Mutations may be detected in a RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a mutated site or that enable amplification of a region containing the mutated site. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a particular mutation. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to identify a mutation in ACTRT1 sequence. Actually numerous strategies for genotype analysis are available (Antonarakis et al, 1989 ; Cooper et al., 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base substitution mutations. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions where the mutations are located. Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides. The mutation may be also detected at a protein level (e.g. for loss of function mutation) according to any appropriate method known in the art. In particular a biological sample, such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of a mutated form of ACTRT1 protein, i.e. antibodies that are capable of distinguishing between a mutated form of ACTRT1 and the wild-type protein, to determine the presence or absence of a ACTRT1 specified by the antibody. The antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies. Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred for they are more reproducible in the long run. Procedures for raising "polyclonal antibodies" are also well known. Alternatively, binding agents other than antibodies may be used for the purpose of the invention. These may be for instance aptamers, which are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. In some embodiments, the first step consists in determining the expression level of
ACTRT1 gene in the biological sample obtained from the subject. Typically, said biological sample is a blood sample or a PBMC sample or is a tissue sample resulting from a biopsy. In some embodiments, the first step consist in i) determining the expression level of ACTRT1 gene, ii) comparing the level determined at i) with a predetermined reference value and iii) concluding that the subject has a ACTRT1 deficiency when the expression level determined at i) is lower than the predetermined reference value. Typically the predetermined reference value is the expression level determined in a healthy population of subject (e.g. the mean expression).
One skilled in the art may easily select the appropriate method for determining the expression level of the gene.
Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trif uoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarnino fluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitro tyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); Ν,Ν,Ν',Ν'-tetramethyl- 6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315- 22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).
In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al, Science 281 :2016- 2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (puhlished May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox- inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).
Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.
Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85 :9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85 :9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467- 1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
It will he appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).
In some embodiments, the nCounter® Analysis system is used to detect intrinsic gene expression. The basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3): 317-325; the contents of which are each incorporated herein by reference in their entireties). The code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed. A pair of probes is designed for each DNA or RNA target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode. This system is also referred to, herein, as the nanoreporter code system. Specific reporter and capture probes are synthesized for each target. The reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over- lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence. Preferably, each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively. The capture probe can comprise a second target-specific sequence; and a first affinity tag. In some embodiments, the capture probe can also comprise one or more label attachment regions. Preferably, the first target- specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected. Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library". The relative abundance of each target is measured in a single multiplexed hybridization reaction. The method comprises contacting the tumor sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex. The complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution. After hybridization, the tripartite hybridized complexes (probe pairs and target) are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes. This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample. All post hybridization steps are handled robotically on a custom liquid-handling robot (Prep Station, NanoString Technologies). Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe,electrophoresed to elongate the reporter probes, and immobilized. After processing, the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies). The expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface. Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance. Data is output in simple spreadsheet format listing the number of counts per target, per sample. This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No. WO 07/076129 and WO07/076132, and US Patent Publication No. 2010/0015607 and 2010/0261026, the contents of which are incorporated herein in their entireties. Further, the term nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No.2010/0047924, incorporated herein by reference in its entirety.
Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources. Other methods for determining the expression level of a gene include the determination of the quantity of proteins encoded by said genes.
Such methods comprise contacting the sample with a binding partner capable of selectively interacting with a marker protein present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. The binding partner may also be an aptamer.
The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
Alternatively an immunohistochemistry (IHC) method may be preferred. IHC specifically provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest. Typically a sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling. Examples of known IHC systems include, for example, EnVision(TM) (DakoCytomation), Powervision(R) (Immunovision, Springdale, AZ), the NBA(TM) kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine(R) (Nichirei Corp, Tokyo, Japan).
In particular embodiment, a tissue section (e.g. a sample comprising cumulus cells) may be mounted on a slide or other support after incubation with antibodies directed against the proteins encoded by the genes of interest. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the proteins of interest.
Therefore IHC samples may include, for instance: (a) preparations comprising cumulus cells (b) fixed and embedded said cells and (c) detecting the proteins of interest in said cells samples. In some embodiments, an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. EXAMPLE:
Cutaneous basal cell carcinoma (BCC) is the most common cancer in Western Countries. It rarely metastases but may cause significant destruction of surrounding tissues. Most BCCs are sporadic but some forms are inherited. Bazex-Dupre-Christol syndrome (BDCS) is an X-linked dominant predisposition to BCCs with typical skin manifestations, suggestive of a primary hair follicle anomaly1'2'3'4. We have mapped the BDCS gene to Xq24- q27 in three large pedigrees5. Studying three additional families, we found genetic homogeneity of the disease (Zmax=15 at Θ = 0) and recombinants allowed to narrow down the genetic interval to 7.5 cM (DXS8057-rs62619090). The encompassed genes (36) were sequenced and an identical single nucleotide insertion in the Actin-Related Protein Tl (Arp- Tl) gene (ACTRTI) was identified in 2/6 families (c.547_548 InsA, Family C and D). This insertion is predicted to cause a frame shift and a premature termination codon at position 199 (p.M183NfsX17). It was found to segregate with the disease in the two families and was absent from available databases (dbSNPs and 1000 genomes) and 600 control chromosomes. ACTRTI expression in HEK293T cells showed that the mutant cDNA encodes a 25 kDa truncated protein. Yet, no mutation of the coding region of ACTRTI was found in the four other families linked to the same interval. No additional exons in ACTRTI were found by 5 '- 3' RACE-PCR and no rearrangements in the candidate region were identified in the remaining 4/6 patients by high-density tiling-path comparative genomic hybridization array.
Because the ACTRTI gene is located in a large 2.5 Mb gene desert, we performed in silico search for highly conserved non-coding elements6 (CNEs), since these elements are known to control expression of neighboring genes7'8'9'10. A total of 17 CNEs were identified. Sanger sequencing detected a g.l27372937A>T variation in CNE12 in two Turkish patients (Family E and F; CNE12 chrX: 127371674-127374249). This variation segregated with the disease and was absent from dbSNP, the 1000 genome databases and from 261 Turkish individuals (404 X chromosomes). This unusually long CNE (2.5 kb) is conserved in mammals (61% identity with the mouse sequence) and teleost fish. In silico predicted secondary structure of CNE12 suggested that it may be transcribed6'11. RT-PCR and in situ hybridization (ISH) experiments showed that CNE 12 was indeed expressed in epidermis and epidermal appendages but not in the dermis. Interestingly, ISH of skin sections showed complete abolition of the specific ncRNA in the two male patients carrying the CNE 12 variation, demonstrating the drastic impact of the g.l27372937A>T variation on its stability or expression. Conversely, ISH analysis detected CNE 12 non-coding RNAs (ncRNAs) in a patient carrying the c.547-548 InsA ACTRTI mutation. Considering that a subset of transcribed ncRNAs act as a novel class of enhancers (eRNAs), we hypothesized that CNE 12 could act as an enhancer in addition to being transcribed. CNE 12 was subcloned in a pGL4.23 lucif erase reporter plasmid. Interestingly, wild-type CNE 12 had an increased enhancer activity in HaCat keratinocyte cell line, while the CNE 12 construct harbouring the A>T mutation was inactive.
Sequence conservation between species is not a consistent indicator of regulatory elements12. Because no mutation in the 17 CNEs surrounding the ACTRTI locus was found in the remaining two families, we carried out systematic array-based capture and high throughput sequencing of the complete 7.5 Mb candidate region in the remaining probands. Owing to the number of variants detected, a specific genome browser and filter was used to detect candidate variants. The ACTRTI mutation and CNE12 variant were confirmed and no mutation in the coding sequences of the other 36 genes located in the 7.5 Mb interval was identified, suggesting that other mutations may involve hitherto unknown ACTRTI regulatory sequences. Two variations were selected (A2: g.l25959394C>G variation and B2: 127968123T>C variation) as candidates and subcloned in pGL4.23. Interestingly, both wild- type sequences had enhancer activity in HaCAT cells but this activity was reduced when the variant constructs were used. Similarly, when used as probes, sequences A2 and B2 stained positively on control skin biopsies, but no staining was detected on skin biopsies from the corresponding patients. Cross tests were positive, demonstrating that absence of ISH staining was patient and mutation-specific.
While epigenetic marks (H3K4mel, H3K36me3 and H3K27ac) usually identify enhancer elements in the genome, no chromatin signature in the vicinity of ACTRTI was found in an atlas of 43,000 candidate active human enhancers13. In order to identify a putative chromatin signature at the ACTRTI locus, chromatin immunoprecipitation (ChIP) followed by quantitative targeted PCR14 was performed on proteins extracted from normal human epidermis. Indeed, an enhancer signature was found for A2, B2 and CNE12 sequences, with an enrichment in H3K27Ac, H3K4Mel . Our results provide genetic and functional evidence that BDCS is caused by loss-of- function mutations altering either the coding region of the ACTRTI gene or eR A enhancers in its transcribed non coding vicinity.
Immuno-histochemical (IHC) analyses in six control skin sections detected Arp-Tl in tissues involved in BDCS (epidermal layers, hair follicles, sebaceous glands and eccrine sweat glands) but not in dermal connective tissue. Interestingly, IHC failed to detect any specific staining in BCC tumors of 9/9 BDCS patients (at least one patient of each BDCS family) and found only a mild signal in unaffected epidermis. Furthermore, Arp-Tl was also undetectable in BCCs from 34/40 unrelated sporadic BCC cases in whom neither germ line nor somatic tumoral ACTRTI mutations had been found. Conversely, Arp-Tl was normally detected in BCCs from two patients with Gorlin syndrome carrying a germline PTCH1 mutation and one Xeroderma Pigmentosum patient. These results emphasize genetic heterogeneity of skin tumors and suggest that sporadic BCCs may quasi-consistently exhibit loss of function at the ACTRTI locus.
Arp-Tl belongs to the Actin-Related Proteins (Arps) family. Nuclear Arps are essential elements of the macromolecular machinery that controls chromatin remodeling, dynamic changes in DNA structure, transcription and re air15'16'17'18'19'20'21-22. Ultra-thin sections of normal skin processed for transmission electronic microscopy analyses detected Arp-Tl in both nucleus and cytoplasm. Subcellular protein fractionation assays showed that Arp-Tl binds chromatin, while the truncated Arp-Tl protein was absent in the chromatin- bound fraction. Arp-Tl chromatin-binding in controls but not BDCS is consistent with its putative involvement in chromatin remodeling and its alteration in the disease.
In order to investigate whether ACTRTI could act as a tumor-suppressor, HEK293T cells were transiently transfected with either wild type or mutant c.547_548 InsA ACTRTI constructs. The wild type but not the mutant construct significantly decreased cell proliferation, by arresting cells in S-phase. We further constructed a MDA-MB231 , invasive hyperproliferative human breast cancer cell line, stably expressing ACTRTI and showed that ACTRTI inhibited in vitro proliferation, by arresting cell cycle in S-phase and migration of MDA-MB231 cells. Moreover, xenograft experiments in NMRI nude mice showed that ACTRTI decreased the development of xenograft tumors and blocked the metastasis effect (n=10 per condition). The sames results were obtained with U20S cells (osteosarcoma).
Since the Hedgehog signaling pathway is activated in more than 70% of BCC cases23'24, we hypothesized that links may exist between Arp-Tl and the Hedgehog pathway. Studying expression of Hedgehog target genes, we found that GLIl, GLI2 and PTCH1 were overexpressed both in non tumoral skin and BCCs derived from BDCS patients carrying either an ACTRTI c.547 548 InsA mutation or a CNE12 variant. Transactivation assay showed that Arp-Tl but not the mutant protein can inhibit the Hedgehog pathway. Consistently, stable expression of ACTRTI significantly reduced the aberrant activation of Hedgehog signaling pathway in MDA-MB231 cells. ChIP assays on control skin revealed that Arp-Tl can bind two regions upstream of the GLIl transcription initiation site, supporting the view that Arp-Tl can directly bind the regulatory sequences of Hedgehog signaling target genes. In keeping with these results, recent studies have shown that GLIl transcriptional activity and Hedgehog signaling are controlled by chromatin regulators, such as Brgl and Snf5, components of the mammalian SWI/SNF chromatin remodeling complex, required for signal-induced transcription of Hedgehog signaling target genes via binding to GLIl regulatory regions. Moreover, loss of Snf5 leads to aberrant activation of the Hedgehog signaling pathway in human rhabdoid tumors25'26. Taken together, our results suggest that transcriptional regulation of GLIl may be pivotal not only for tumor suppressor activity of Snf5 and Brgl, but for Arp-Tl as well.
Germline mutations in the ACTRTI gene and its non-coding surrounding elements in BCCs represent a hitherto unreported mechanism of inherited predisposition to skin tumors. This study highlights the impact of intergenic mutations in human diseases. Elucidating the disease mechanism in BDCS, a rare inherited condition, has thus shed light on a most common human cancer, BCC. Developing novel Hedgehog signaling inhibitors targeting Arp- Tl will hopefully help improving treatment of this frequent, potentially devastating condition. REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
1 Bazex, A., Dupre, A., and Christol, B., [Follicular atrophoderma, baso-cellular proliferations and hypotrichosis]. Ann Dermatol Syphiligr (Paris) 93 (3), 241 (1966).
2 Plosila, M., Kiistala, R., and Niemi, K. M., The Bazex syndrome: follicular atrophoderma with multiple basal cell carcinomas, hypotrichosis and hypohidrosis. Clin Exp
Dermatol 6 (1), 31 (1981).
3 Rapelanoro, R., Taieb, A., and Lacombe, D., Congenital hypotrichosis and milia: report of a large family suggesting X-linked dominant inheritance. Am J Med Genet 52
(4), 487 (1994).
4 Herges, A., Stieler, W., and Stadler, R., [Bazex-Dupre-Christol syndrome. Follicular atrophoderma, multiple basal cell carcinomas and hypotrichosis]. Hautarzt 44 (6), 385 (1993).
5 Vabres, P. et al, The gene for Bazex-Dupre-Christol syndrome maps to chromosome Xq. J Invest Dermatol 105 (1), 87 (1995).
6 Nobrega, M. A., Ovcharenko, I., Afzal, V., and Rubin, E. M., Scanning human gene deserts for long-range enhancers. Science 302 (5644), 413 (2003).
7 Oram, U. A. et al., Long noncoding RNAs with enhancer-like function in human cells. Cell 143 (1), 46.
8 Oram, U. A., Derrien, T., Guigo, R., and Shiekhattar, R., Long noncoding RNAs as enhancers of gene expression. Cold Spring Harb Symp Quant Biol 75, 325.
9 Oram, U. A. and Shiekhattar, R., Noncoding RNAs and enhancers: complications of a long-distance relationship. Trends Genet 27 (10), 433.
10 Oram, U. A. and Shiekhattar, R., Long non-coding RNAs and enhancers. Curr Opin Genet Dev 21 (2), 194.
11 Gruber, A. R. et al, The Vienna RNA websuite. Nucleic Acids Res 36 (Web Server issue), W70 (2008). 12 Fisher, S. et al, Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312 (5771), 276 (2006).
13 Andersson, R. et al, An atlas of active enhancers across human cell types and tissues. Nature 507 (7493), 455.
14 Visel, A. et al, ChlP-seq accurately predicts tissue-specific activity of enhancers. Nature 457 (7231), 854 (2009).
15 Heid, H. et al, Novel actin-related proteins Arp-Tl and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head. Exp Cell Res 279 (2), 177 (2002).
16 Kuroda, Y. et al, Brain-specific expression of the nuclear actin-related protein ArpNalpha and its involvement in mammalian SWI/SNF chromatin remodeling complex.
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17 Georgieva, M., Harata, M., and Miloshev, G., The nuclear actin-related protein Act3p/Arp4 influences yeast cell shape and bulk chromatin organization. J Cell Biochem 104 (1), 59 (2008).
18 Gerhold, C. B. et al, Structure of Actin-related protein 8 and its contribution to nucleosome binding. Nucleic Acids Res 40 (21), 11036.
19 Oma, Y. and Harata, M., Actin-related proteins localized in the nucleus: from discovery to novel roles in nuclear organization. Nucleus 2 (1), 38.
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Claims

CLAIMS:
1. A method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an Arp-Tl polypeptide (SEQ ID NO: 1) or a functional variant or fragment thereof.
2. The method of claim 1 wherein the functional variant of the Arp-Tl polypeptide has at least 70% of identity with SEQ ID NO: 1.
3. The method of claim 1 wherein the fragment comprises 10; 1 1; 12; 13; 14; 14; 15; 16;
17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51 ; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82;
83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103;
104; 105; 106; 107; 108; 109; 110; 111 ; 112; 113; 114; 115; 1 16; 1 17; 1 18; 1 19; 120;
121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133; 134; 135; 136; 137;
138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161 ; 162; 163; 164; 165; 166; 167; 168; 169; 170; 171 ;
172; 173; 174; 175; 176; 177; 178; 179; 180; 181 ; 182; 183; 184; 185; 186; 187; 188;
189; 190; 191; 192; 193; 194; 195; 196; 197; 198; 199; 200; 201; 202; 203; 204; 205;
206; 207; 208; 209; 210; 211 ; 212; 213; 214; 215; 216; 217; 218; 219; 220; 221; 222;
223; 224; 225; 226; 227; 228; 229; 230; 231; 232; 233; 234; 235; 236; 237; 238; 239; 240; 241; 242; 243; 244; 245; 246; 247; 248; 249; 250; 251; 252; 253; 254; 255; 256;
257; 258; 259; 260; 261; 262; 263; 264; 265; 266; 267; 268; 269; 270; 271; 272; 273;
274; 275; 276; 277; 278; 279; 280; 281; 282; 283; 284; 285; 286; 287; 288; 289; 290;
291; 292; 293; 294; 295; 296; 297; 298; 299; 300; 301; 302; 303; 304; 305; 306; 307;
308; 309; 310; 311; 312; 313; 314; 315; 316; 317; 318; 319; 320; 321; 322; 323; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341 ;
342; 343; 344; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 357; 358;
359; 360; 361; 362; 363; 364; 365; 366; 367; 368; 369; 370; 371 ; 372; 373; 374 or 375 consecutive amino acids of SEQ ID NO: 1.
4. A method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a nucleic acid encoding for an Arp- Tl polypeptide (SEQ ID NO: l) or a functional variant or fragment thereof.
5. The method of claim 4 wherein the nucleic acid molecule is SEQ ID NO:2 or a nucleic acid sequence having at least 70% of identity with SEQ ID NO:2.
6. The method of claim 4 wherein the nucleic acid molecule is incorporated in a vector.
7. The method of claim 1 or 4 wherein the subject suffers from a cancer selected from the group consisting of cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; muco epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangio sarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglio neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
8. A method for screening a plurality of candidate compounds useful for treating cancer comprising the steps consisting of (a) testing each of the candidate compounds for its ability to enhancing ACTRT1 gene expression and (b) and positively selecting the candidate compounds capable of enhancing said expression.
9. A method for determining whether a subject has a or is at risk of having cancer comprising the steps consisting of detecting an ACTRTl deficiency in the biological sample obtained from the subject and concluding that the subject has a cancer or is at risk of having a cancer when an ACTRTl deficiency is detected.
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EP1900824A1 (en) * 2006-09-14 2008-03-19 Deutsches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts Gene expression signature for the prognosis, diagnosis and therapy of prostate cancer and uses thereof
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