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WO2011068865A1 - Le micro-arn-101 stimule la croissance indépendante des œstrogènes et confère une résistance au tamoxifène dans des cellules cancéreuses er-positives - Google Patents

Le micro-arn-101 stimule la croissance indépendante des œstrogènes et confère une résistance au tamoxifène dans des cellules cancéreuses er-positives Download PDF

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WO2011068865A1
WO2011068865A1 PCT/US2010/058551 US2010058551W WO2011068865A1 WO 2011068865 A1 WO2011068865 A1 WO 2011068865A1 US 2010058551 W US2010058551 W US 2010058551W WO 2011068865 A1 WO2011068865 A1 WO 2011068865A1
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mir
cancer cells
antisense
rna
cells
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Yin-Yuan Mo
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Southern Illinois University System
Southern Illinois University Edwardsville
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • MicroRNAs are a class of naturally occurring small non-coding RNAs that are capable of regulating expression of protein-coding genes [Ambros, (2004) Nature, 431:350- 355]. They exert their silencing function usually by binding to the 3 ' untranslated region (3'- UTR) of a target gene with partial sequence homology, leading to post-transcriptional repression. Because of this unique feature, a single microRNA (miR) can have multiple targets [Brennecke et al., (2005) PLoSBiol, 3:e85] and thus, microRNAs can regulate a large number of protein-coding genes and impact a variety of cellular pathways.
  • microRNAs play a role in developmental timing, cell death, cell proliferation, hematopoiesis and patterning of the nervous system and oncogenesis [Calin et al., (2006) Nat Rev Cancer, 6:857-866]. Now it becomes evident that microRNAs play a broader role in regulation of cellular functions including estrogen signaling and tamoxifen resistance [Maillot et al., (2009) Cancer Res, 69:8332-8340].
  • ER cytoplasmic estrogen receptors
  • miR-206 is down-regulated in ER positive MCF-7 cells (ATCC HTB-22; differentiated human mammary epithelium including ability to process estradiol via cytoplasmic estrogen receptors), and ectopic expression; i.e., gene expression in a tissue in which the gene is normally not expressed, of miR-206 can suppress ER expression and cell growth [Adams et al., (2007) Mol Endocrinol, 21:1132-1147; Kondo et al., (2008) Cancer Res, 68:5004-5008].
  • miR-221/222 have also been implicated in tamoxifen resistance through a similar mechanism [Miller et al., (2008) J Biol Chem, 283:29897-29903; Zhao et al., (2008) J Biol Chem, 283:31079-31086]. More recently, miR-22 was reported to be the most prominent microRNA for ER because miR-22 is able to suppress ER at a greater degree than other known ER-targeting microRNAs such as 221/222 or miR-206 [Pandey et al., (2009) Mol Cell Biol, 29:3783-3790].
  • tamoxifen resistance associated with suppression of ER may provide an explanation as to why some breast tumors fail to respond to tamoxifen therapy; however, little is known as to whether microRNAs play any role in intrinsic resistance to tamoxifen in ER positive tumors.
  • the invention provides methods for assaying estrogen receptor-positive sample cancer cells for resistance to tamoxifen therapy by assaying said cancer cells for the overproduction of miR-101 comprising: measuring the level of miR-101 in the sample cancer cells, measuring the level of miR-101 in control cancer cells known to be tamoxifen sensitive, and comparing the miR-101 levels. miR-101 overproduction is indicated if the miR-101 level in the sample cancer cells is significantly higher than that in the control cancer cells.
  • the cancer cells are from a breast cancer.
  • the invention provides methods for inhibiting the phosphorylation of PTEN in estrogen receptor-positive cancer cells comprising inhibiting the activity of miR-101.
  • the activity of miR-101 is inhibited by providing said cells an antisense miR-101 composition.
  • the invention provides methods for inhibiting the phosphorylation of Akt in estrogen receptor-positive cancer cells comprising inhibiting the activity of miR-101.
  • the activity of miR-101 is inhibited by providing said cells an antisense miR-101 composition.
  • the invention provides methods for increasing the sensitivity to tamoxifen of tamoxifen-resistant estrogen receptor-positive cancer cells in an animal comprising providing those cells with an antisense miR-101 composition.
  • methods in accordance with the invention include administering to the animal one or more agents or compounds selected from the group consisting of a radionuclide, cancer chemotherapeutic agent, targeted anticancer agent, DNA interacalating/damaging agent, cell cycle check point inhibitor, antimetabolites, HSP inhibitor, antibiotic, kinase inhibitor, radionuclide, biologically active polypeptide, antibody, lectin, toxin, hormone, matrix metalloproteinase inhibitor, angiostatic steroid or combinations thereof, in addition to the antisense miR-101 composition.
  • agents or compounds selected from the group consisting of a radionuclide, cancer chemotherapeutic agent, targeted anticancer agent, DNA interacalating/damaging agent, cell cycle check point inhibitor, antimetabolites, HSP inhibitor, antibiotic, kina
  • a method in accordance with the invention include administering to the animal one or more agents or compounds selected from the group consisting of 13 II, 90Y, 11 lln, 211 At, 32P, genistein, adriamycin, ansamycin, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, an epothilone, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine,
  • mercaptopurine meplhalan, methotrexate, rapamycin, sirolimus, mitomycin, mitotane, mitoxantrone, nitrosurea, pamidronate, pentostatin, plicamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, taxanes, vinblastine, vincristine, vinorelbine, taxol, combretastatins, discodermolides, transplatinum, bleomycin, hormones, tamoxifen, diethylstilbestrol, axitinib, avastin, marimastat, bevacizumab, carboxyamidotriazole, TNP- 470, CM101, IFN-a, EL- 12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, cartilage
  • 2-methoxyestradiol 2-methoxyestradiol, tecogalan, thrombospondin, prolactin, ⁇ 3 inhibitor, tecogalan, BAY 12-9566, AG3340, CGS27023A, COL-3, vitaxin, ZD0101, TNP-40, thalidomide, squalamine, IM862, PTK787, fumagillin, an analogue of fumagillin, BB-94, BB-2516 linomid, oxaliplatin, paclitaxel and combinations thereof, in addition to the antisense miR- 101 composition.
  • the invention also provides a pharmaceutical composition that comprises a therapeutically effective amount of an antisense miR-101 molecule dissolved or dispersed in a pharmaceutically acceptable carrier.
  • the invention provides antisense miR- 101 compositions comprising one or more of a DNA, RNA, LNA or PNA molecule of 10 to 30 bases that is complementary to a portion of SEQ ID NO: 12, wherein the in vitro administration of the antisense miR-101 composition to a plurality of breast cancer cells reduces the level of RNA comprising SEQ ED NO: 12 in those cells by at least 50% or inhibits the activity of RNA comprising SEQ ID NO: 12 in those cells by at least 50%.
  • FIG. 1 is in five parts.
  • FIG. 1A is a schematic that illustrates preparation of a microRNA library used for in vivo selection study.
  • a microRNA precursor containing a DNA fragment (about 500 bp) was cloned under the control of a CMV promoter of a lentiviral vector, pCDH-CMV-MCS-EFl-copGFP (System Biosciences, Mountain View, CA).
  • PI and P4 are the primers derived from the vector and used to recover microRNA precursors from the cells or tumors.
  • P2 and P3 were used to clone microRNA precursors into the lentiviral vector.
  • FIG. IB is a schematic showing the progression of in vivo selection studies in which MCF-7 cells were infected with either the library pool or vector control, and then injected orthotopically into the fat pads of female nude mice. Tumors were only produced via library pool injection, and the tumors so produced were profiled to determine mRNA enrichment, and the enriched mRNAs were reintroduced into MCF-7 cells.
  • FIG. IB is a schematic showing the progression of in vivo selection studies in which MCF-7 cells were infected with either the library pool or vector control, and then injected orthotopically into the fat pads of female nude mice. Tumors were only produced via library pool injection, and the tumors so produced were profiled to determine mRNA enrichment, and the enriched mRNAs were reintroduced into MCF-7 cells.
  • FIG. 1C is a graph that illustrates the relative growth of the MCF-7 cells infected with the microRNA library before in vivo selection (BIVS) or after in vivo selection (AIVS) in E2-free medium for one, ten and twenty days (Dl, D10 and D20, respectively). Relative cell growth was determined by the trypan blue exclusion method.
  • FIG. ID illustrates that the ATVS cells (upper line) are more tamoxifen resistant than the BIVS cells. Cytotoxicity assays were performed using the standard MTT method at the indicated concentrations of tamoxifen for four days after addition of the drug.
  • FIG. ID illustrates that the ATVS cells (upper line) are more tamoxifen resistant than the BIVS cells. Cytotoxicity assays were performed using the standard MTT method at the indicated concentrations of tamoxifen for four days after addition of the drug.
  • IE contains western blots that illustrate the effect of microRNAs on estrogen receptor (ER), Akt and pAkt, with a ⁇ -actin as a control.
  • ER estrogen receptor
  • Akt Akt
  • pAkt pAkt
  • FIG. 2 illustrates that miR-101 promotes estrogen independency and tamoxifen resistance.
  • FIG. 2A is a graph showing relative cell growth versus tamoxifen concentration for MCF-7 cells infected with vector (pCDH), miR-101 or miR-380 (negative microRNA control).
  • FIG. 2A illustrates that miR-101 confers tamoxifen resistance (upper line).
  • FIG. 2B shows that miR- 101 activates pAkt. Infected cells were grown in E2-free medium for two days before harvesting for western blot.
  • FIG. 2C shows that miR-101 has no effect on ERa expression. MCF-7 cells infected with vector control, miR-22 or miR-101 were used for western blot. miR-22 serves as a positive control.
  • FIG. 2D shows a series of bar graphs of luciferase assays with the reporter carrying three copies of estrogen response elements (3 x ERE-Luc).
  • MCF-7 cells were transiently transfected with the 3 x ERELuc along with pCDH (vector), miR-22 or miR-101. Luciferase assays were carried out 24 hours after transfection. Values are mean ⁇ SE of three independent studies; **, p ⁇ 0.01.
  • FIG. 3 illustrates miR-101 -mediated Akt activation.
  • FIG. 3 A is a bar graph that shows relative growth of MCF-7 cells infected with either vector control or miR-101 (hatched) that were grown in normal medium for four days before harvesting for measuring the number of viable cells by the trypan blue staining method. The relative cell growth at day 4 was compared to the number at day 0, which was normalized to one.
  • FIG. 3B shows the results when the same infected MCF-7 cells were cultured in E2-free medium for the indicated number of days. Note that miR-101 suppressed cell growth in the normal medium, but promoted cell growth in the E2-free medium.
  • FIG. 3 A is a bar graph that shows relative growth of MCF-7 cells infected with either vector control or miR-101 (hatched) that were grown in normal medium for four days before harvesting for measuring the number of viable cells by the trypan blue staining method. The relative cell growth at day 4 was compared to the number at day 0, which was normalized to one.
  • FIG. 3C contains a series of western blots that illustrate that miR-101 causes Akt activation.
  • MCF-7 cells infected with the vector control, miR-380 (negative control) or miR-101 were split and grown either in normal medium or E2-free medium for two days.
  • FIG. 3D in two panels, contains bar graphs that show the relative pAkt level (miR-101 vs vector) in cells grown in different media, normal (dark bar) or E2-free (light bar) as a ratio of pAkt/Akt (FIG. 3D-1) or relative pAkt (FIG. 3D- 2).
  • FIG. 3D contains bar graphs that show the relative pAkt level (miR-101 vs vector) in cells grown in different media, normal (dark bar) or E2-free (light bar) as a ratio of pAkt/Akt (FIG. 3D-1) or relative pAkt (FIG. 3D- 2).
  • 3E contains western blots that illustrate that miR-101-mediated Akt activation can be blocked by antisense miR-101 (anti-miR-101) oligonucleotide.
  • MCF-7 cells infected with either vector control or miR-101 were transiently transfected with antisense miR-101. The cells were then split and cultured in either normal medium or E2-free medium for two days before harvesting for western blot.
  • Values in FIG. 3A, FIG. 3B and FIG. 3D are means ⁇ SE of three independent studies; **, p ⁇ 0.01 ; *, p ⁇ 0.05.
  • FIG. 4 shows that MAGI-2 (a membrane-associated guanylate kinases family member) is a direct target for miR-101.
  • FIG. 4A contains a series of bar graphs that show that miR-101 suppresses the luciferase activity of the Luc-MAGI2-UTR reporter.
  • MCF-7 cells were co-transfected with Luc-MAGI2-UTR reporter or a mutated Luc-MAGI2-UTR (Luc-MAGI2-UTR-m; hatched) reporter and vector control or miRlOl, and luciferase assays were carried out 24 hours after transfection.
  • FIG. 4B illustrates suppression of the endogenous MAGI-2 protein as detected by western blot.
  • MCF-7 cells were transiently transfected either with a scrambled oligonucleotide or antisense miR- 101 (anti-miR- 101) and harvested for western blot two days after transfection. The exposure times were different for these two blots to reveal the relative levels of MAGI-2 between vector and miR-101 or scrambled and antisense miR-101.
  • MCF-7 cells were transfected with Myc-tagged MAGI-2 expression vector and harvested for western blot two days after transfection.
  • FIG. 4C-2 stable MCF-7 transductants with commercially available MAGI-2 shRNA were harvested, and western blot was performed to determine the levels of MAGI-2 and Akt protein expression.
  • MAGI-2 shRNA-1 (sh#l) caused obvious suppression of MAGI-2, whereas MAGI-2 shRNA-2 (sh#2) had little effect on MAGI-2 expression.
  • pAkt was increased by MAGI-2 shRNA-1 (sh#l), but not by MAGI-2 shRNA-2 (sh#2).
  • FIG. 4D illustrates that MAGI-2 shRNA-1 confers tamoxifen resistance.
  • Stable MCF-7 clones carrying either the vector control or MAGI-2 shRNAs were treated with tamoxifen (TAM, hatched; 2 ⁇ ) or the DMSO-containing medium without tamoxifen (DMSO) for four days, and the relative cell number was determined by trypan blue staining. Values are mean ⁇ SE of three independent studies; **, p ⁇ 0.01; *, p ⁇ 0.05.
  • FIG. 5 in two panels, illustrates the effects of miR-101 on pAkt and p-PTEN.
  • FIG. 5 A MCF-7 cells infected with either the vector control or miR-101 were transiently transfected with antisense miR-101 (anti-miR-101) or a scrambled oligonucleotide. The cell culture was then split and portions of the cells were cultured in either normal medium or E2- free medium for two days before harvesting for western blot.
  • FIG. 5B MCF-7 cells infected with either the vector control or miR-101 were transiently transfected with RFP vector or PTEN-RFP. Western blot was carried out same as in FIG. 5A.
  • FIG. 6 in two parts, shows in FIG. 6A that the six identified microRNAs from the library of 331 microRNAs conferred tamoxifen resistance to infected MCF-7 cells, whereas FIG. 6 B illustrates that miR-101 also activates Akt in BT474 cells (ATCC HTB-20), a line of human breast tumor cells that supports mouse mammary tumor virus replication.
  • FIG. 7 show a series of western blots in which expression of the proteins shown on the right are seen to be the same in cells infected (transfected) with vector alone or with miR-101.
  • the present invention contemplates methods that utilize the discovery that the over-production (or over-expression) of the microRNA referred to in the art as miR-101 provides estrogen-independent growth as well as resistance to growth inhibition by tamoxifen to certain cancer cells, such as breast cancer cells, that contain the cytoplasmic estrogen receptor (ER) in usual amounts for the cell type from which the cancer developed.
  • miR-101 provides estrogen-independent growth as well as resistance to growth inhibition by tamoxifen to certain cancer cells, such as breast cancer cells, that contain the cytoplasmic estrogen receptor (ER) in usual amounts for the cell type from which the cancer developed.
  • ER cytoplasmic estrogen receptor
  • the present invention contemplates a method for assaying estrogen receptor-positive cancer cells for resistance to tamoxifen therapy that comprises assaying those cancer cells for over-production of miR-101. That over-production can be assayed in many ways.
  • Estrogen receptor-positive (ER) cancer cells usually proliferate when cultured in a usual mammalian cell culture medium that contains added estrogen.
  • over-production is assayed by culturing the cells in the absence of added estrogen (17- ⁇ - estradiol or E2) and in the presence of an effective amount of antisense miR-101
  • oligonucleotide whereby non-proliferation of the cells indicates over-production of miR- 101.
  • over-production is assayed by culturing the cancer cells in the presence of an estrogen-free medium, whereby proliferation of those cells indicates overproduction of miR-101.
  • Another embodiment contemplates assay of over-production of miR- 101 by culturing the cancer cells in the presence of an estrogen-free medium and assaying for the presence of an increased amount of pAkt in the cancer cells, relative to the amount of pAkt normally present in cancer cells that do not over-produce pAkt.
  • the over-production is assayed by culturing the cancer cells and assaying for the presence of an increased amount of pPTEN in those cells relative to the amount of pPTEN normally present in such cells.
  • assay methods is exemplified hereinafter.
  • Another aspect contemplates a method of inhibiting the phosphorylation of PTEN in estrogen receptor-positive cancer cells that comprises inhibiting the activity of miR-101.
  • the activity of miR-101 is inhibited by transfecting those cells with an antisense miR-101 composition.
  • a further aspect of the invention contemplates a method of inhibiting the phosphorylation of Akt in estrogen receptor-positive cancer cells that comprises inhibiting the activity of miR-101.
  • the activity of miR-101 is inhibited by transfecting those cells with an antisense miR-101 composition.
  • a still further aspect of the invention contemplates a method of increasing the sensitivity of tamoxifen-resistant estrogen receptor-positive cancer cells to tamoxifen that comprises transfecting those cells with an antisense miR-101 composition.
  • One benefit is the identification of a biological marker, miR-101, that when over- produced in estrogen receptor-containing cancer cells such as breast cancer cells, identifies cells that are resistant to growth inhibition by tamoxifen and exhibit estrogen-independent growth.
  • An advantage of the invention is that one can assay estrogen receptor-positive cancer cells for resistance to tamoxifen therapy by assaying those cancer cells for over- production of miR- 101.
  • a further benefit of the invention is that miR- 101 over-production can be assayed in many ways.
  • a further advantage of the invention is that one can increase the sensitivity to tamoxifen of tamoxifen-resistant estrogen receptor-positive cancer cells by transfecting those cells with an antisense miR-101 composition.
  • the term "providing” refers making a molecule available to cells in vivo or in vitro, so that the molecule can act biologically on the cells, e.g., a vector which is taken up and expressed or a naked RNA which enters the cell and acts as an antisense molecule.
  • an antisense miR-101 refers to a result which substantially decreases the level or expression of, including for example, an about 20% reduction, preferably an about 25% reduction, more preferably an about 33% reduction, even more preferably an about 50% reduction, even more preferably an about 67% reduction, even more preferably an about 80% reduction, even more preferably an about 90% reduction, even more preferably an about 95% reduction, even more preferably an about 99% reduction, and most preferable complete silencing of miR-101.
  • an "effective amount” or “therapeutic effective amount” can refer to a substantial decrease in the level or functional activity of miR-101 as compared to a negative control, either in an unaffected tissue from the subject being treated, or in a suitable corresponding tissue of an unaffected control animal. It can be convenient to refer to the decrease in miR-101 in a test sample in terms of its proportion to the negative control.
  • the miR-101 level in the test sample can be decreased by an amount equal to the level of the negative control. More preferably, the test sample level can be decreased by an amount equal to about twice the level of the negative control (a two fold reduction).
  • the level can be decreased by about five fold, about six fold, about seven fold, about eight fold, about nine fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 1000 fold, or even about 10,000 fold as compared to the level of the negative control.
  • complete silencing of miR-101 is most preferred.
  • nucleic acid or “oligonucleotide” refers to multiple nucleotides (i.e., molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g.
  • a substituted pyrimidine e.g. cytosine (C), thymidine (T) or uracil (U)
  • purine e.g.
  • nucleic acid also encompasses nucleic acids with substitutions or
  • nucleic acid includes DNAs, RNAs and peptide nucleic acids (“PNAs").
  • isolated nucleic acid refers primarily to a RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) can further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • isolated RNA includes preparing RNA in a histologic section for detection by in situ hybridization.
  • polypeptide and “polypeptide” are used interchangeably herein and refer to a compound made up of a chain of amino acid residues linked by peptide bonds.
  • An "active portion" of a polypeptide means a peptide that is less than the full length polypeptide, but which retains measurable biological activity and retains biological detection.
  • Locked nucleic acids are a class of nucleic acid analogues in which the ribose ring is "locked” by, e.g., a methylene bridge connecting the 2'-0 atom and the 4'-C atom.
  • LNA nucleosides contain the common nucleobases (T, C, G, A, U and mC) and are able to form base pairs according to standard Watson-Crick base pairing rules. However, by "locking" the molecule with the methylene bridge the LNA is constrained in the ideal conformation for Watson-Crick binding. When incorporated into a DNA oligonucleotide, LNA therefore makes the pairing with a complementary nucleotide strand more rapid and increases the stability of the resulting duplex.
  • a "LNA/DNA mixmer” or “mixmer” is used to refer to a nucleic acid that contains at least one LNA unit and at least one RNA or DNA unit (e.g., a naturally-occurring RNA or DNA unit).
  • a "headmer” is defined by a contiguous stretch of beta-D-oxy-LNA or LNA derivatives at the 5'-end followed by a contiguous stretch of DNA or modified monomers recognizable and cleavable by the RNaseH towards the 3'-end
  • a "tailmer” is defined by a contiguous stretch of DNA or modified monomers recognizable and cleavable by the RNaseH at the 5'-end followed by a contiguous stretch of beta-D-oxy-LNA or LNA derivatives towards the 3 '-end.
  • a "gapmer” is based on a central stretch of 4-15 base DNA (gap) typically flanked by 1 to 6 residues of 2'-0 modified nucleotides (beta-D-oxy-LNA in our case, flanks) which are able to act via an RNaseH mediated mechanism to reduce the target sequence's level.
  • said sub-sequence comprises a stretch of 3 to about 5 nucleotide analogues, such as LNA nucleotide analogues, as defined herein, followed by a stretch of 4 to about 15 nucleotides, which is followed by a stretch of 3 to about 5 nucleotide analogues, such as LNA nucleotide analogues as defined herein, optionally with a single nucleotide at the 3' end.
  • LNA nucleotide analogues such as LNA nucleotide analogues
  • said sub-sequence comprises a stretch of 4 nucleotide analogues, such as LNA nucleotide analogues, as defined herein, followed by a stretch of 8 nucleotides, which is followed by a stretch of 3 nucleotide analogues, such as LNA nucleotide analogues as defined herein, optionally with a single nucleotide at the 3' end.
  • 4 nucleotide analogues such as LNA nucleotide analogues, as defined herein
  • 8 nucleotides which is followed by a stretch of 3 nucleotide analogues, such as LNA nucleotide analogues as defined herein, optionally with a single nucleotide at the 3' end.
  • conjugate refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the delivery, trafficking, detection or isolation of that sequence.
  • the conjugate is cholesterol added to the 3' end of an LNA, which confers the ability of the LNA of the invention to be cell-permeable.
  • histidine residues e.g., 4 to 8 consecutive histidine residues
  • amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) can be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography.
  • tag moieties are known to, and may be envisioned by, the skilled artisan, and are contemplated to be within the scope of this definition.
  • a “pharmaceutical formulation” includes formulations for human and veterinary use with no significant adverse toxicological effect.
  • a “pharmaceutically acceptable formulation” as used herein refers to a composition or formulation that permits the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
  • the phrase "significantly higher" used herein in relation to overproduction of miR-101 in estrogen receptor-positive sample cancer cells is used to mean that one can see a difference in miR-101 between cells in an assayed sample and cells of a control. That difference can be determined by standard assays such as in situ hybridization, northern blot, blot hybridization, PCR, TMA, invader or microarray; as well as by culturing the sample cancer cells in the absence of estrogen, whereby proliferation indicates overproduction of miR-101; by culturing the sample cancer cells in the absence of estrogen and the presence of an effective amount of antisense miR-101, whereby non-proliferation indicates
  • co-administration and “combination therapy” refer to administering to a subject two or more therapeutically active agents.
  • the agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulation and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be coadministered.
  • Nucleic acid sequences that are embodiments of the invention and for uses in accordance with the invention include:
  • MAGI2-UTR-ml-3.1 Anti-Sense Primer SEQ ID NO: 4
  • miPv-101 SEQ ID NO: 12
  • Complementary nucleic acids have the potential to base pair.
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the invention provides miR- 101 and antisense miR- 101 (anti-miR- 101) compositions comprising one or more of a DNA, RNA, LNA or PNA molecule having a length of 10 nucleotides to about 170 nucleotides, preferably 10 to about 30 nucleotides in length, more preferably about 18 to about 25 nucleotides in length, which is complementary to a portion of SEQ ID NO: 12, e.g., comprising SEQ ID NOs: 13 or 14, wherein the in vitro administration of the antisense miR-101 composition to a cell reduces the level of RNA comprising SEQ ID NO: 12 in that cell by at least about 50%, preferably by at least about 80%, more preferably by at least about 90% and most preferably by at least about 95% or inhibits the activity comprising RNA SEQ ID NO: 12 in that cell by at least about 50%), preferably by at least about 80%, more preferably by at least about 90% and most preferably by at least about 95%
  • compositions that comprise an effective amount of an antisense miR-101 molecule dissolved or dispersed in a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers include those that are aqueous and are liquids at room temperature.
  • An antisense miR-101 molecule in accordance with the invention includes one that contains one or more pseudophosphate bonds at one or both of the 3'- and 5 '-termini, and preferably a plurality of pseudophosphate bonds at each of the 3'- and 5 '-termini.
  • Such antisense miR-101 molecules can be in the form of a pri-miRNA, pre-miRNA, mature miR A, ds miRNA and fragments or variants thereof.
  • the antisense miR-101 molecule is a naked synthetic RNA.
  • the antisense miR-101 molecule is a synthetic, chemically modified RNA or DNA that has been modified with a chemical moiety selected from the group consisting of a boranophosphate, 2'-0-methyl, 2'-fluoro, PEG, terminal inverted-dT base, and combinations thereof and/or modified to include a 2'-F, 2'-ara-F (FANA), 4'-thioribose, 2'-OMe, 2'-MOE, NMAC, DMAEAc, LNA, ENA, 5-propynyl-C, G- Clamp, 5-methyl-C, MMI or phosphorothioate chemical structure.
  • a chemical moiety selected from the group consisting of a boranophosphate, 2'-0-methyl, 2'-fluoro, PEG, terminal inverted-dT base, and combinations thereof and/or modified to include a 2'-F, 2'-ara-F (FANA), 4'-thioribose, 2'-OMe,
  • the invention provides antisense miR-101 molecules that comprise the nucleic acid sequences of SEQ ID NO: 13 or 14, as well as a DNA, RNA, LNA or PNA molecule of 10 to 30 bases that is complementary to a portion of SEQ ED NO: 12, wherein the in vitro administration of the antisense miR-101 composition to a cell reduces the level of miR-101 in that cell by at least 50% or inhibits the activity of miR-101 in that cell by at least 50%.
  • a contemplated antisense nucleic acid in accordance with the invention is e.g., 10 nucleotides to about 170 nucleotides in length, preferably 18 to about 25 nucleotides in length.
  • Suitable oligonucleotides useful in various embodiments of the present invention can be composed of naturally occurring nucleobases, sugars and internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions that function similarly or with specific improved functions. Fully or partly modified or substituted oligonucleotides are often preferred over native forms because of several desirable properties of such oligonucleotides, for instance, the ability to penetrate a cell membrane, good resistance to extra- and intracellular nucleases, high affinity and specificity for the nucleic acid target.
  • Suitable oligonucleotides can be unmodified or chemically modified single- stranded DNA molecules. Suitable oligonucleotides are from about 10 to about 30 bases in length, preferably from about 18 to about 25 bases in length and hybridize under stringent conditions (e.g., high salt, high temperature) to provide a desired sequence, e.g., SEQ ID NO: 12. Other embodiments include, e.g., the sponge oligonucleotide of SEQ ID NO: 14 is a suitable antisense approach.
  • a contemplated miR- 101 or antisense miR- 101 molecule is preferably an oligonucleotide that contains one to more preferably up to about 8 non-phosphodiester bonds between (linking) the nucleotides. Such non-phosphodiester bonds are preferably arrayed at both the 5'- and the 3 '-termini of the molecule. Contemplated non-phosphodiester bonds are referred to herein as pseudophosphate bonds, and are resistant to exonuclease and/or endonuclease action as compared to phosphodiester bonds.
  • pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate,
  • exonuclease-and/or endonuclease-resistant oligonucleotides can be obtained by blocking the 3'- and/or 5'-terminal nucleotides with substituent groups such as acridine, caps such as 5-methylguanosine or poly(A) tails, as are well known in the art.
  • LNA 2'-4'-linked (Locked Nucleic Acid; LNA) modification
  • LNA modification is deemed herein to be a
  • LNA modification is also deemed to embrace both methylene and ethylene linkages between the 2'- and 4'-positions of the sugar ring of a nucleotide base.
  • That LNA modification is also particularly preferred to be present at least between the two terminal bases at each of the 3'- and 5 '-termini of an antisense miR-101 or miR-101 molecule, and there are more preferably four bases so linked at each terminus. It is to be understood that LNA modification can be used along with phosphodiester bonds as well as non-phosphodiester bonds between the bases.
  • LNA phosphorarnidite monomers are available from Exiquon and also from Glen Research Corp. of Sterling, VA.
  • Suitable miR-101 and antisense miR-101 molecules include those in which one or more of the bases is modified, e.g., to include a 2'-F, 2'-ara-F, FANA (2'-fluoro-P-D- arabinonucleic acid), 4'-thioribose, 2'-OMe (i.e., 2 -O-methyl backbone), 2'-MOE (2'-0- dimethylaminoethyloxyethyl-modified), NMAC (N-methylacetamide), DMAEAc
  • miR-101 sense and antisense molecules in accordance with the invention include those wherein the molecule comprises, e.g., a gapmer, mixmer or headmer.
  • a miR-101 and antisense miR-101 composition in accordance with the invention can further comprise a pharmaceutically-acceptable carrier.
  • the miR-101 and antisense miR-101 molecules provided by the invention include those where at least one of said one or more DNA, RNA , LNA or PNA oligonucleotides is modified by the addition of any one of cholesterol, bis-cholesterol, PEG, PEG-ylated carbon nanotube, poly-L- lysine, cyclodextran, polyethylenimine polymer or peptide moieties.
  • oligonucleotides in accordance with the invention can be modified by any polymeric species including a synthetic or naturally occurring polymer or protein.
  • Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone.
  • An artificial or synthetic polynucleotide is any polynucleotide that is polymerized in vitro or in a cell free system and contains the same or similar bases but can contain a backbone of a type other than the natural ribose-phosphate backbone. These backbones include: PNAs
  • peptide nucleic acids peptide nucleic acids
  • PS oligonucleotide phosphorothioates
  • phosphorodiamidates morpholinos, and other variants of the phosphate backbone of native nucleic acids. See, Dias et al., (2002) Mol Cancer Ther, 1:347-355; and Sahu et al., (2007) Cur Pharma
  • Bases include any of the known base analogs of DNA and RNA, as well as purines and pyrimidines, the latter further including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.
  • Synthetic derivatives of purines and pyrimidines include, but are not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • Modifications in accordance with the invention also include methylphosphonate oligonucleotides that are noncharged oligomers, in which a nonbridging oxygen atom is replaced by a methyl group at each phosphorus in the oligonucleotide chain.
  • the phosphorothioates in the phosphorothioate diastereomer have improved nuclease stability.
  • a preferred embodiment involves the replacement of the hydrogen at the 2'-position of ribose by an O-alkyl group, most frequently methyl.
  • Suitable oligonucleotides also include embodiments that do not possess the natural phosphate-ribose backbone.
  • PNAs Peptide Nucleic Acids
  • PNAs are nucleic acid analogues that contain an uncharged, flexible, polyamide backbone comprised of repeating N-(2- aminoethyl)glycine units to which the nucleobases are attached via methylene carbonyl linkers. These oligomers can form very stable duplexes or triplexes with nucleic acids: single or double-strand DNA or RNA. The property of high-affinity nucleic acid binding can be explained by the lack of electrostatic repulsion because of the absence of negative charges on the PNA oligomers.
  • PNAs are not substrates for the RNase H or other RNases, the antisense mechanism of PNAs depends on steric hindrance. PNAs also can bind to DNA and inhibit RNA polymerase initiation and elongation, as well as the binding and action of transcription factors, such as nuclear factor ⁇ . PNAs also can bind mRNA and inhibit splicing or translation initiation and elongation.
  • Phosphorodiamidate morpholino oligomers in which the deoxyribose moiety is replaced by a morpholine ring and the charged phosphodiester intersubunit ' linkage is replaced by an uncharged phosphorodiamidate linkage, also are suitable for use in accordance with the invention. These oligonucleotides are very stable in biological systems and exhibit efficient antisense activity in cell-free translation systems and in a few cultured animal cell lines.
  • oligonucleotide is the N3' ⁇ P5' PN, which result from the replacement of the oxygen at the 3' position on ribose by an amine group.
  • N3' ⁇ P5' PN which result from the replacement of the oxygen at the 3' position on ribose by an amine group.
  • These oligonucleotides can, relative to their isosequential phosphodiester counterparts, form very stable complexes with RNA and single- or double-stranded DNA. Specificity, as well as efficacy, can be increased by using a chimeric oligonucleotide, in which the RNase
  • H-competent segment usually a phosphorothioate moiety
  • a phosphorothioate moiety is bounded on one or both termini by a higher-affinity region of modified RNA , e.g., a 2'O-alkyloligoribonucleotides. This substitution not only increases the affinity of the oligonucleotide for its target, but also reduces the cleavage of nontargeted mRNAs by RNase H.
  • LNA Locked Nucleic Acid
  • the LNA units comprise at least one beta-D-oxy-LNA unit(s) such as 4, 5, 6, 7, 8, 9, or 10 beta-D-oxy-LNA units.
  • All the LNA units can be, e.g., beta-D-oxy- LNA units, although it is considered that the oligomeric compounds, such as the antisense oligonucleotide, can comprise more than one type of LNA unit.
  • the oligomeric compound can comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, ena-LNA and/or alpha-LNA in either the D-beta or L-alpha configurations or combinations thereof.
  • Embodiments of the invention can comprise nucleotide analogues, such as LNA nucleotide analogues, which typically comprise a stretch of 2-6 nucleotide analogues, such as LNA nucleotide analogues, as defined herein, followed by a stretch of 4-12 nucleotides, which is followed by a stretch of 2-6 nucleotide analogues, such as LNA nucleotide analogues, as defined herein.
  • the oligonucleotides of the present invention can comprise modified bases such that the oligonucleotides retain their ability to bind other nucleic acid sequences, but are unable to associate significantly with proteins such as the RNA degradation machinery.
  • the oligonucleotide agents featured in the invention also may include 2'-0-methyl, 3'-0- pixyl, 5'-0- pixyl, 2'-fluorine, 2'-0-methoxyethyl, 2'-0- aminopropyl, 2'-amino, and/or phosphorothioate linkages and the like.
  • ENAS ethylene nucleic acids
  • 2'-4'-ethylene-bridged nucleic acids e.g., 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, also may increase binding affinity to the target.
  • nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, also may increase binding affinity to the target.
  • the invention provides a method for assaying estrogen receptor-positive sample cancer cells for resistance to tamoxifen therapy by assaying those cancer cells for the overproduction of miR-101 comprising: measuring the level of miR-101 in the sample cancer cells, measuring the level of miR-101 in control cancer cells known to be tamoxifen sensitive, and comparing the levels of miR-101. miR-101 overproduction is indicated if the level of miR-101 in the cancer cells is significantly higher than that in the control cancer cells.
  • Methods in accordance with the invention include, e.g., wherein the
  • overproduction is assayed by culturing said cells in the absence of estrogen and the presence of an effective amount of antisense miR-101, whereby non-proliferation indicates overproduction of miR-101; by culturing the cancer cells in the absence of estrogen, whereby proliferation indicates overproduction of miR-101; by culturing the cancer cells in the absence of estrogen (in an estrogen-free medium) and assaying for the presence of an increased amount of pAkt in the cells; and by culturing the cancer cells in the absence of estrogen and assaying for the presence of an increased amount of pPTEN in said cells.
  • the invention provides a method of inhibiting the phosphorylation of Akt in estrogen receptor-positive cancer cells or the phosphorylation of PTEN in estrogen receptor- positive cancer cells that comprises inhibiting the activity of miR-101, including wherein the activity of miR-101 is inhibited by providing those cells an antisense miR-101 composition.
  • the antisense miR-101 composition can comprise a vector expressing an antisense miR-101 RNA or the antisense miR-101 composition can comprise an antisense miR-101 RNA, DNA or PNA, including, e.g., a modified RNA, DNA or PNA molecule.
  • Methods in accordance with the invention include those wherein the antisense miR-101 composition is provided to the cancer cells in vitro or in vivo and whether the cells are from a breast cancer or other tumor, e.g., an ovarian or endometrial cancer.
  • the invention provides a method for increasing the sensitivity to tamoxifen of tamoxifen-resistant estrogen receptor-positive cancer cells in an animal that comprises providing the cells with an antisense miR-101 composition.
  • An antisense miR-101 composition in accordance with the invention include that wherein a vector expresses the antisense miR-101 RNA, as well as where the antisense miR-101 composition comprises an antisense miR-101 RNA, DNA or PNA.
  • a method for increasing the sensitivity to tamoxifen of animal cells in accordance with the invention includes, e.g., administering to the animal one or more compounds ( or agents) selected from the group consisting of a radionuclide, cancer chemotherapeutic agent, targeted anticancer agent, DNA interacalating/damaging agent, cell cycle check point inhibitor, anti-metabolite, HSP inhibitor, antibiotic, kinase inhibitor, radionuclide, biologically active polypeptide, antibody, lectin, toxin, hormone, matrix metalloproteinase inhibitor, angiostatic steroid and combinations thereof, as well as administering to the animal one or more compounds (or agents) selected from the group consisting of 1311, 90Y, 11 lln, 21 lAt, 32P, genistein, adriamycin, ansamycin, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine
  • a pharmaceutical composition (or formulation) in accordance with the invention can be, e.g., carried in a liposome, polymer-based nanoparticle, cholesterol conjugate, cyclodextran complex, polyethylenimine polymer or a protein complex.
  • a pharmaceutical composition in accordance with the invention can be, e.g., formulated administration directly to diseased tissue, intravenously, subcutaneously, intramuscularly, nasally, intraperitonealy, vaginally, anally, orally, intraocularly or intrathecally.
  • a composition provided by the invention includes one modified to facilitate cell- type selective targeting or to enhance the uptake of oligonucleotides.
  • CTLs include lipoprotein receptors (particularly those in the liver), integrins and receptor tyrosine kinases.
  • Suitable CPPs include polycationic peptides rich in arginine and lysine and membrane-interactive hydrophobic sequences, e.g., KALA peptide
  • GR KRRQRRRPPQ SEQ ID NO: 18
  • Penetratin GenBank Accession No. 1KZ0_A. See also, Juliano et al., (2008) Nucleic Acids Research, 36(12):4158-4171 and Yu et al., (2009) AAPS Journal, ll(l):195-203.
  • the invention provides methods that contemplate transfecting cancer cells, those cancer cells can be transfected in vitro, and the cancer cells are preferably breast cancer cells.
  • Suitably methods of in vitro transfection are well known and are exemplified hereinafter. Among those illustrative methods is transfection by infection of the target cell with a lentiviral vector or an adenoviral vector.
  • Suitable methods of in vivo transfection are also well known in the art and can also be used for in vitro transfection.
  • Illustrative examples include delivery using liposomes or nanoparticles, in particular albumin bound nanoparticles, coupling to peptides or stabilizing the oligonucleotides by chemical modification such as 2'O-methylation or 2'-4' linkage of the sugar phosphate backbone.
  • Use of the 2'-4'-linked (Locked Nucleic Acid; LNA) modification available from Santaris Pharma of Denmark and Enzon Pharmaceuticals of New Jersey, has been shown to increase the affinity of a miR molecule to mRNA or miRNA, and to confer resistance to exonuclease activity.
  • LNA Locked Nucleic Acid
  • Each of the various species of nucleic acids provided by the invention can be targeted to tumor cells with a monoclonal antibody or affinity peptide.
  • RNAfectinTM Transfection Reagent an aqueous lipid-based liquid composition available from QIAGEN, Inc., Valencia CA, for transforming cells with antisense miR-101.
  • Antisense miR-101 is administered to cells in an effective amount, e.g., about 100 nM per 70% confluent cells in a 6- or 12-well plate.
  • In vitro lentiviral infections are typically carried out at about multiplicity of infection of about 1 to about 3.
  • the targeting portion is an analog of an oligonucleotide wherein at least one of the 2'-deoxy ribofuranosyl moieties of the nucleoside unit is modified.
  • a hydrogen or a hydroxyl, halo, azido, amino, methoxy or alkyl functional group may be added.
  • H OH, lower alkyl, substituted lower alkyl, F, CI, Br, CN, CF3, OCF3, OCN, 0-alkyl, S-alkyl, SOME, S0 2 Me, ON0 2 , N0 2 , N 3 , NH 2 , NH-alkyl, OCH 2 CH 2 , OCH 2 CCH, -OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, aminoalkylamino, heterocycloalkylamino, polyalkylamino, substituted silyl, a RNA cleaving moiety or a group for improving the pharmacodynamic properties of an oligonucleotide or a group for improving the pharmacokinetic properties of an oligonucleotide where alkyl is a straight or branched chain of d to Q 2 , can be used, with unsaturation within the carbon chain, such as allyloxy, being particularly preferred.
  • Suitable modifications of the miR-101 and anti- miR-101 oligonucleotides in accordance with the invention include those resulting in cyclobutyl moieties, e.g., cyclobutyl moieties that include a purine or pyrimidine heterocyclic base attached at a C-l cyclobutyl position and where cyclobutyl moieties are joined through a C-2, C-3 or C-4 position on a first of said cyclobutyl moieties and a C-2, C-3, or C-4 position on a second of the cyclobutyl moieties by a linkage that contains 4 or 5 consecutive, covalently-bound atoms selected from the group consisting of C, N, O, S, Si and P.
  • miR- 101 and anti- miR- 101 oligonucleotides in accordance with the present invention include those resulting in a compound based on a N-2 substituted purine, a 3-deazapurine ring system, and a compound with purines comprising a tether portion and at least one reactive or non-reactive functionality.
  • such embodiments include nucleosides, nucleoside analogs, nucleotides, nucleotide analogs, heterocyclic bases, and heterocyclic base analogs based on the purine ring system.
  • Methods for assaying for the amount of miR-101 produced are also well known to those skilled in this art.
  • assay techniques include western blots, cell growth, and luciferase activity after transfection.
  • suitable assays include, e.g., in situ hybridization, northern blot, dot blot, microarray, RT-PCR, invader, TMA, and mass spectroscopy.
  • a pharmaceutical composition or formulation is also contemplated. Such a composition is typically utilized in a contemplated method as discussed herein.
  • a contemplated pharmaceutical composition that comprises an effective amount of antisense miR-101 molecule dissolved or dispersed in a pharmaceutically acceptable carrier.
  • a useful pharmaceutically acceptable carrier is well known in the art and is typically aqueous and is a liquid at room temperature.
  • nucleic acids administered in vivo are taken up and distributed to cells and tissues [Huang, et al., (2004) FEBS Lett. 558(l-3):69-73].
  • Nyce et al. have shown that antisense oligodeoxynucleotides (ODNs) when inhaled bind to endogenous surfactant (a lipid produced by lung cells) and are taken up by lung cells without a need for additional carrier lipids [Nyce et al., (1997) Nature, 385:721-725].
  • siRNAs have been used for therapeutic silencing of an endogenous genes by systemic administration
  • the physician evaluates circulating plasma levels, formulation toxicities, and progression of the disease.
  • the dose administered to a 70 kilogram patient is typically in the range equivalent to dosages of currently-used therapeutic antisense oligonucleotides such as Vitravene.RTM. (fomivirsen sodium injection) which is approved by the FDA for treatment of cytomegalo viral RNA, adjusted for the altered activity or serum half-life of the relevant composition.
  • the carrier is preferably adapted for parenteral administration as compared to oral or topical administration.
  • pharmaceutically acceptable salts, buffers and the like are present that collectively are referred to as pharmaceutically acceptable diluents as compared to those that can be present in a composition that is not intended for pharmaceutical use, as in an in vitro assay.
  • a contemplated miR-101 or antisense miR-101 molecule is a polyphosphate ester that is anionic at physiological pH values and is usually present in a contemplated pharmaceutical composition as a salt of a cation such as sodium, potassium, magnesium or ammonium ions, or mixtures of such cations. Further pharmaceutically acceptable cations can be found in Berge, (1977) J. Pharm. Sci. 68(1): 1 19 for lists of commonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds.
  • the antisense miR-101 molecule can be present as part of a vector such as a lentivector discussed elsewhere, or as a nucleic acid free of a vector (as an oligonucleotide).
  • a contemplated pharmaceutical composition can be administered in a formulation containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • parenteral as used herein includes subcutaneous injections, intravenous (which is most preferred), intramuscular, intrasternal injection, or infusion techniques.
  • Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, Pennsylvania; 1975 and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms. Marcel Decker, New York, N.Y., 1980.
  • a preferred injectable preparation for example, sterile injectable aqueous solution or suspension can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • suitable dispersing or wetting agents and suspending agents include water, Ringer's solution, and isotonic sodium chloride solution, phosphate- buffered saline.
  • a contemplated antisense miR-101 molecule is provided as a dry powder, typically a lyophilized powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.
  • a sterile solution can be prepared by dissolving the antisense miR-101 molecule in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile antisense miR-101 molecule in a previously sterilized solvent under sterile conditions.
  • a mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a contemplated compound is administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.
  • a sample to be assayed such as cells and tissue can be used.
  • These in vitro compositions typically contain the water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0 -8.5, preferably about pH 7.2-7.4, depending on the assay to be performed, as is well known.
  • the pharmaceutical composition is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active compound.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules.
  • ERa, EGFR and p-EGFR, HER2 and p-HER2, PTEN and p- PTEN were purchased from Epitomics (Burlingame, CA); Akt, pAkt, mTOR and CTMP were purchased from Cell Signaling Technologies (Danvers, MA); MAGI-2 and ⁇ -actin were purchased from Sigma-Aldrich (St. Louis, MO); and R-Ras was purchased from Santa Cruz (Santa Cruz, CA).
  • Secondary antibodies conjugated with IRDye 800CW or IRDye 680 were purchased from LICOR Biosciences (Lincoln, NE).
  • PCR primers (hereinafter) and antisense miR-101 LNA oligo were purchased from IDT (Coralville, IA). Tamoxifen was purchased from Sigma-Aldrich. MAGI-2 shRNAs were purchased from Open Biosystems (Huntsville, AL).
  • HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Cambrex) supplemented with 10% FBS. All media contained 2 mM glutamine, 100 units of penicillin/ml, and 100 ⁇ g of streptomycin/ml. Cells were incubated at 37°C and supplemented with 5% C02 in a humidified chamber.
  • ATCC American Tissue Culture Collection
  • FBS foetal bovine serum
  • HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Cambrex) supplemented with 10% FBS. All media contained 2 mM glutamine, 100 units of penicillin/ml, and 100 ⁇ g of streptomycin/ml. Cells were incubated at 37°C and supplemented with 5% C02 in a humidified chamber.
  • DMEM Dulbecco's modified Eagle's medium
  • pre-microRNA expression vectors an about 0.5-kb DNA fragment covering a pre-microRNA was first amplified, using human genomic DNA (Promega, Madison, WI) as a template. PCR reactions were performed using the high fidelity Phusion enzyme (New England Biolabs, Ipswich, MA) and corresponding specific primers. The amplified fragment was cloned into a PCR cloning vector (pCR8), and subsequently cloned into a lentiviral vector (pCDH-CMV-MCS-EFl-copGFP from System Biosciences, Mountain View, CA) at EcoRl and Notl sites. Where there was an internal EcoRl or Notl site, partial digestions were performed to obtain the DNA fragment carrying the pre-microRNA.
  • pCR8 PCR cloning vector
  • pCDH-CMV-MCS-EFl-copGFP from System Biosciences, Mountain View, CA
  • microRNA library used in this study consisted of 331 microRNA precursors (System Biosciences).
  • MCF-7 cells were transfected with antisense miR- 101 using the RNAfectin reagent (Applied Biological Materials) following the manufacturer's protocol. In brief, cells were seeded at 40% confluence in either 12- or 6-well plates, then transfected with 100 nM of oligos in serum-free medium when the cells reached about 70% confluence. The serum-free medium was then replaced by normal growth medium after 16 hours of transfection. Stable transductants were also prepared for those microR As enriched from the microRNA library by infection as described in the following section.
  • HEK293T cells were transfected using the calcium phosphate method, as described previously (Sachdeva et al., (2009) Proc Natl Acad Sci USA, 106:3207-3212). The transfected cells were grown overnight (about 18 hours) before being harvested and lysed for use in a luciferase assay, protein extraction or RNA detection.
  • Lentiviral packaging was carried out in HEK293T cells using a packaging system from System Biosciences per the manufacturer's protocol, as described previously [Sachdeva et al., (2009) Proc Natl Acad Sci USA, 106:3207-3212].
  • a packaging system from System Biosciences per the manufacturer's protocol, as described previously [Sachdeva et al., (2009) Proc Natl Acad Sci USA, 106:3207-3212].
  • the microRNA library pool a large-scale preparation was carried out, and the viral particles were concentrated by centrifugation. The final titer was at least 1x108.
  • MCF-7 cells were mixed with viral particles in the presence of polybrene (0.8 ⁇ g/ml) in a 6-well plate at a multiplicity of infection (MOI) of about 1 to about 3.
  • MOI multiplicity of infection
  • the Myc-tagged MAGI-2 construct was a generous gift from Dr. Yutaka Hata (Tokyo Medical and Dental University, Tokyo, Japan).
  • the luciferase-UTR reporter constructs were prepared by introducing the MAGI-2 3'-UTR carrying a putative miR-101 binding site into pGL3 control vector (Promega).
  • the 3'-UTR sequence was amplified by PCR using the primers MAGI2-UTR-PGL3C-Xbal-5.1 and MAGI2-UTR-PGL3C-Xba 1-3.1 (hereinafter) and the resulting clone was directly cloned into the Xbal site of the pGL3 control vector (Promega, Madison, WI) using the Cold Fusion cloning kit (System
  • the UTR was amplified by using the primers MAGI2-UTR-m 1-5.1 (hereinafter) and MAGI2-UTR- ml-3.1 for site 1; MAGI2-UTR-m2-5.1 and MAGI2-UTR-m2-3.1 for site 2; and MAGI2- UTR-m3-5.1 and MAGI2-UTR-m3-3.1 for site 3.
  • the same cloning strategy was used to clone those sequences into the pGL3 control vector at the Xbal site.
  • PTEN-coding region was amplified using the primers PTEN-RED-Not 1-5.1 and PTEN-RED-Not 1-3.1. That product was then directly cloned into a modified pCMV-RFP at the Notl site. PCR products were verified by DNA sequencing.
  • Luciferase assays for the UTR reporters were carried out as previously described (Sachdeva et al., (2009) Proc Natl Acad Sci USA, 106:3207-3212). First, cells were transfected with the appropriate plasmids in 12-well plates. Then, the cells were harvested and lysed for a luciferase assay 24 hours after transfection. Luciferase assays were performed using a luciferase assay kit (Promega) according to the manufacturer's protocol. Renilla luciferase was used for normalization. To determine the effect of miR-101 on ERE- luciferase activity, the assays were performed in MCF-7 cells infected with miR-101 using the standard method.
  • PCR reactions were performed to amplify the MAGI-2 3'-UTR according to the standard three-step procedure. Annealing temperature varied depending on the primers used.
  • RTPCR total RNA was isolated using the Trizol reagent (Invitrogen) per the
  • MCF-7 cells infected with either the vector control or the microRNA library pool were cultured in growth medium to the exponential stage before harvesting for injection.
  • Female athymic nude (nu/nu) mice (4-5 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were maintained in the Southern Illinois University School of Medicine's accredited animal facility.
  • 1.5 x 106 cells in 0.1 ml PBS containing 50% matrigel were injected into the mammary pads of mice. The animals were sacrificed, and their tumors were harvested and weighed 6 to 7 weeks after injection.
  • This Example demonstrates the enrichment of microRNAs required for estrogen independent-growth by in vivo selection.
  • a library of 331 human microRNA precursors was prepared from System Biosciences, and cloned under a CMV promoter of a lentiviral vector, pCDH-CMV-MCS-EFl-copGFP (System Biosciences, Mountain View, CA) (FIG. 1 A). Each of these clones carried an approximately 500 base pair (bp) genomic DNA fragment containing a microRNA precursor in the middle. Real-time RT- PCR was used for following the expression of corresponding mature microRNAs was demonstrated.
  • FIG. IB is a schematic flow chart for in vivo selection of microRNAs required for estrogen-independent growth.
  • MCF-7 cells were first infected with this library as a pool. Next, infected cells were injected into mammary fat pads of female nude mice without estradiol. This is a selection process because only those microRNAs that can activate estrogen-independent growth were of interest.
  • microRNAs are enriched in this mixed population through growth of tumors.
  • those cells infected with vector control or non- relevant microRNAs are not able to grow tumors.
  • Tumors were formed from MCF-7 cells infected with the library (FIG. 1C), but no tumor was formed from MCF-7 cells infected with vector control.
  • Cells were recovered from these tumors by cell culture, all of which showed GFP positive (not shown), indicating that microR As are stably maintained in the cells. These recovered cells were named ATVS (After In Vivo Selection). Infected cells prior to selection are referred to as BIVS (Before In Vivo Selection).
  • miR-101 is shown as capable of promoting estrogen-independent growth and conferring tamoxifen resistance without impacting ER expression or ER activity, implying that dysregulation of miR-101 in tumors can lead to intrinsic resistance to tamoxifen regardless of ER status.
  • One of the notable molecular alterations caused by miR-101 is upregulation of pAkt. This miR-101 -mediated activation of Akt can be blocked by antisense miR-101, suggesting the specific effect of miR- 101.
  • microRNAs were responsible for estrogen-independent growth and tamoxifen resistance.
  • genomic DNA was isolated from the tumors as well as RNA.
  • MicroRNA expression was profiled by real time PCR against the microRNA library using the tumor genomic DNA as a template.
  • Six microRNAs (miR-101, miR-140, miR-380, miR- 552, miR-578 and miR-599) were identified that were enriched through this approach (data not shown).
  • Real time RT-PCR also confirmed the increased levels of these six microRNAs (data not shown).
  • miR-101 was the microRNA that promoted the highest degree of estrogen independent growth.
  • miR-101 was able to confer estrogen- independent growth and tamoxifen resistance; however, miR-380 did not have this capability (FIG. 2A and 2B).
  • the miR-101 cells expressed a higher level of pAkt than either the vector control or miR-380 (FIG. 2B).
  • miR-101 also activated Akt in another ER-positive breast cancer cell line, BT474 (FIG. 6B).
  • miR-101 did not affect ER expression as determined by western blot (FIG. 2C) or its activity as shown by luciferase reporter carrying 3 copies of estrogen response elements (3 x ERE-Luc) (FIG. 2D). These results indicate a novel mechanism for miR-101 to cause estrogen-independency and tamoxifen resistance.
  • miR-101 had been shown to be a tumor suppressor capable of inhibiting cell growth [Friedman et al., (2009) Cancer Res, 69:2623-2629; Varambally et al., (2008) Science, 322:1695-1699].
  • cell proliferation assays also indicated that miR-101 had a negative effect on cell growth in normal E2 -containing medium (FIG. 3A).
  • E2-free medium miR-101 promoted cell growth and proliferation (FIG. 3B). Consistent with this result, tumors derived from the miR-101 cells were found to be smaller than those from the vector control cells when the mice were implanted with 17- ⁇ - estradiol pellets.
  • miR-101 was suppressed using an antisense miR-101 LNA (locked nucleic acid) oligonucleotide.
  • antisense miR- 101 inhibited miR- 101 -induced Akt activation in both normal and E2-free medium (FIG. 3E), suggesting a specific miR-101 effect on pAkt.
  • PI3K inhibitor (LY29) was able to enhance the sensitivity to tamoxifen in the miR-101 cells.
  • PI3K/Akt is a key factor in the mitogen- mediated cell growth and proliferation pathway.
  • Many factors are known to regulate pAkt. The most important and direct upstream factor is PTEN, a well-known inhibitor of PI3K. Suppression of PTEN expression or reduction of its activity increases pAkt levels.
  • Western blot analysis revealed only a slight reduction of the PTEN levels in the miR-101 cells when compared to the vector control cells.
  • mTOR which, together with Rictor, forms the mTORC2 complex and phosphorylates Akt, activating mTORCl through PRAS40 suppression [Kim et al., (2002) Cell, 110:163-175).
  • An increase in the level of mTOR expression would result in Akt activation; however, no difference in mTOR expression levels between the vector control and miR-101 (FIG. 7) was detected.
  • Targetscan [Grimson et al., (2007) Mol Cell, 27:91-105] and MicroCosm Targets [Griffiths- Jones et al., (2008) Nucleic Acids Res, 36:D154-158], CTMP (carboxy-terminal modulator protein, also called thioesterase superfamily member 4), MAGI-1 and MAGI-2 were found to carry potential miR-101 binding sites at their 3'-UTRs.
  • CTMP carboxy-terminal modulator protein, also called thioesterase superfamily member 4
  • MAGI-1 and MAGI-2 were found to carry potential miR-101 binding sites at their 3'-UTRs.
  • CTMP is a negative Akt regulator that works by dephosphorylating pAkt (Maira et al., (2001) Science, 294:374-380].
  • luciferase assays using a reporter carrying the CTMP 3'- UTR and western blot indicated that miR-101 had no effect on CTMP.
  • MAGI proteins are members of a family of membrane-associated guanylate kinases
  • Luciferase reporter assays revealed that miR-101 caused a slight reduction of Luc-MAGI-1- UTR activity.
  • miR-101 significantly suppressed Luc-MAGI-2-UTR activity (FIG. 4A), likely through direct interactions with the miR-101 binding sites in the MAGI-2 UTR, because mutations at these sites reversed its suppression. Furthermore, miR-101 also suppressed the endogenous MAGI-2 protein levels (FIG. 4B). Real time RT-PCR showed no significant effect of miR-101 on MAGI-2 mRNA expression, suggesting that this miR- 101- mediated suppression of MAGI-2 takes place through translational repression. [0170] To determine the effect of MAGI-2 on Akt, ectopic expression of MAGI-2 was able to reduce the pAkt level (FIG. 4C-1). In contrast, suppression of MAGI-2 by RNAi increased pAkt, mimicking the miR-101 action on pAkt (FIG. 4C-2).
  • MAGI-2 shRNA-1 promoted MCF-7 cells' survival in E2-free medium.
  • Stable MCF-7 clones carrying either the vector control or MAGI-2 shRNAs were cultured in E2-free medium, and images were taken at day 10 (not shown).
  • MAGI-2 shRNA-1 promoted estrogen-independent growth, mimicking the effect of miR- 101.
  • MAGI-2 interacts with the C-terminus of PTEN through its PDZ domain [Tolkacheva et al., (2001) Cancer Res, 61:4985-4989] such that PTEN is able to convert PIP3 to PIP2 [Engelman (2009) Nat Rev Cancer, 9:550-562].
  • the vector control cells or the miR-101 cells were transfected with scrambled oligo or antisense miR-101 and then cultured in normal or E2 ⁇ free medium for two days.
  • miR-101 increased the pAkt level in both the normal and the E2-free medium (FIG. 5A); again, the Akt activation by miR-101 was higher in the E2-free medium than in normal medium.
  • miR-101 suppressed MAGI-2 expression, especially in E2-free medium.
  • p-PTEN was increased, whereas the total PTEN level remained almost the same for both the vector control and miR-101.
  • PTEN was also ectopically expressed. As shown FIG. 5B, this expression caused a reduction of pAkt in the vector control cells; in contrast, it had little effect on pAkt expression in the miR-101 cells, indicating that miR-101 blocks the PTEN-mediated inhibition of Akt and miR-101 targets such as MAGI-2 are required for this process.

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Abstract

Dans un aspect, la présente invention concerne un procédé pour analyser des cellules cancéreuses positives au récepteur d'œstrogène (ER) pour déterminer la résistance à une thérapie avec le tamoxifène qui comprend l'analyse de ces cellules cancéreuses pour la surproduction de miR-101. Un autre aspect concerne un procédé d'inhibition de la phosphorylation de PTEN dans des cellules cancéreuses positives au récepteur d'œstrogène qui comprend l'inhibition de l'activité de miR-101. Un autre aspect de l'invention concerne un procédé d'inhibition de la phosphorylation de Akt dans des cellules cancéreuses positives au récepteur d'œstrogène qui comprend l'inhibition de l'activité de miR-101. Un autre aspect supplémentaire de l'invention concerne un procédé d'augmentation de la sensibilité au tamoxifène des cellules cancéreuses positives au récepteur d'œstrogène résistantes au tamoxifène qui comprend la transfection de ces cellules avec miR-101 antisens. La présente invention concerne en outre des compositions pharmaceutiques comprenant des molécules miR-101 antisens.
PCT/US2010/058551 2009-12-01 2010-12-01 Le micro-arn-101 stimule la croissance indépendante des œstrogènes et confère une résistance au tamoxifène dans des cellules cancéreuses er-positives Ceased WO2011068865A1 (fr)

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Cited By (1)

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US20110224149A1 (en) * 2010-02-23 2011-09-15 Xiao Gary Guishan Polynucleotides for use in treating and diagnosing cancers

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