WO2010100328A1 - NUCLEIC ACIDS REGULATING OESTROGEN RECEPTOR (ER)-α SIGNALING IN BREAST CANCER - Google Patents

Abstract

This invention relates to use of a nucleic acid or nucleic acids, preferably mi RNAs disclosed for the manufacture of a medicine for the treatment of breast cancer. This method also relates to a method of obtaining a marker value or values useful for differentiating patients with an oestrogen dependent breast cancer with an increased risk of progression into an oestrogen independent breast cancer by determining from a sample or samples of a tumour of breast cancer mi RNAs disclosed and establishing a marker value and/or values, which are a determined amount or amounts as such, or a function of the determined amount or amounts of disclosed mi RNAs. This invention also relates to a method of treatment and/or prevention of breast cancer.

Description

NUCLEIC ACIDS REGULATING OESTROGEN RECEPTOR (ER)-α SIGNALING IN BREAST CANCER

FIELD OF THE INVENTION

This invention relates to the use of nucleic acids for the manufacture of a medicine for the treatment of breast cancer. This invention also relates to a method of obtaining marker values useful for differentiating patients at risk of progression of an oestrogen dependent breast cancer into an oestrogen independent breast cancer. This invention further relates to a method for the treatment of oestrogen dependent breast cancer and/or prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer. This invention relates to particular miRNAs essential for the different aspects of the invention.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

Oestrogen receptor (ER) status is important for the development, progression, and treatment of breast cancer, and it has become a major target for the treatment of this disease. The two human oestrogen receptors, ERa and ERβ, are encoded by separate ESR1 and ESR2 genes, respectively [Cheskis BJ et al. J Cell Physiol 2007 213(3):610-7; Hewitt SC et al. Reproduction 2003 125(2):143-9]. ERa is the primary mediator of the mitogenic activities of oestrogen in breast tissue, and its levels are tightly controlled in a tissue- and development-specific manner [Couse JF and Korach KS Endocr Rev 1999 20(3):358-417]. Understanding the molecular basis of ER expression is critically important as most breast cancers express high levels of ERa, and the inhibition of oestrogenic signals by antioestrogens and other endocrine therapies is part of the routine management of breast cancer patients. However, many patients relapse during endocrine therapies and develop oestrogen-independent breast cancer [Milano A et al. Eur J Cancer 2006 42(16):2692-705].

Multiple mechanisms are known to regulate ERa levels in cells, including transcriptional control [Reid G et al. Cell MoI Life Sci 2002; 59(5):821 -31 ], rate of mRNA degradation [Kenealy MR et al. Endocrinology 2000 141 (8):2805-13] and, according to recent evidence, microRNAs (miRNAs) [Adams BD et al. MoI Endocrinol 2007 21 (5):1 132-47; Zhao JJ et al. J Biol Chem 2008 283(45): 31079- 31086]. MiRNAs target messenger RNAs (mRNAs) for cleavage or translational repression by binding to the 3' untranslated region (3' UTR) of the target mRNAs [Esquela-Kerscher A and Slack FJ. Nat Rev Cancer 2006 6(4):259-69]. Genes encoding 721 miRNAs have so far been identified (miRBase v.14.0) [Griffiths- Jones S et al. Nucleic Acids Res 2008 36(Database issue):D154-8], and they are predicted to regulate the expression of at least 60 % of all human protein-encoding genes [Friedman RC et al. Genome Res 2009 19:92-105]. Several studies have linked miRNAs in regulating breast cancer progression and hormone receptor status [Verghese ET et al. J Pathol 2008 215(3):214-21 ]. A few miRNAs have been associated with breast cancer proliferation and invasion [Huang Q et al. Nat Cell Biol 2008 10(2):202-10; Ma L, Teruya-Feldstein J and Weinberg RA Nature 2008 455(7210):256; Tavazoie SF et al. Nature 2008 451 (7175):147-52], and miR- 7, miR-128a, miR-210, and miR-516-3p are involved with the aggressiveness of lymph node-negative, oestrogen receptor-positive breast cancer [Foekens JA et al. Proc Natl Acad Sci U S A 2008; 105(35):13021 -6]. MiRNA expression profiles can distinguish different breast cancer subtypes and classify oestrogen receptor status [Mattie MD et al. MoI Cancer 2006; 5:24; Blenkiron C et al. Genome Biol 2007; 8(10):R214]. For instance, the expression of miR-142-5p, miR-200a, miR-205 and miR-25 positively correlates with the ERa expression [Mattie MD et al. (2006)]. Adams et al. (2007) showed how miR-206 binds to two sites in the 3'UTR region of ERa, and directly downregulates ERa expression [Adams BD et al. 2007]. In addition, low miR-206 expression has been linked to ERα-positive clinical breast cancers [Kondo N et al. Cancer Res 2008; 68(13):5004-8]. A recent study also described miR-221/222 as negatively regulating ERa and contributing to the Tamoxifen resistance in breast cancer [Zhao JJ et al. (2008)]. Liu et al. (Gastroenterology 2009 136: 683-693) identifies miR-18a to be differentially expressed between male and female hepatocellular carcinoma (HCC), and to target the ESR1 gene leading to downregulation of ERa protein. miR-18a overexpression had opposite effects on cell proliferation between the hepatoma and breast cancer cell lines: miR-18a-mediated downregulation of ERa increased cell proliferation in HCC cells but decreased the proliferation of MCF-7 breast cancer cells.

WO 2008/014008 discloses a method of reducing angiogenesis, the method comprising contacting a cell with an effective amount of an inhibitory nucleic acid molecule complementary to at least a portion of a microRNA nucleic acid molecule of the mir-17-92 cluster, thereby reducing angiogenesis. WO 2008/014008 identifies mir-18a as an alternative of six microRNAs as well as treating breast cancer as an alternative of seventeen cancers to be treated.

WO 2009/004632 discloses a method of treating a hyperproliferative or degenarative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide agent capable of down-regulating at least one microRNA selected from the group wherein miR-18a and miR-18b are defined within the group comprising 19 alternatives altogether.

WO 2009/004632 further discloses a pharmaceutical composition comprising at least one microRNA selected from the group wherein miR-18a and miR-18b are defined within the group comprising 16 alternatives altogether.

WO 2007/016548 discloses methods and composition for the diagnosis, prognosis and treatment of breast cancer and provides methods of identifying anti-breast cancer agents. Methods of diagnosis, prognosis of breast cancer as well as identification of anti-breast cancer agents relate to measurement of a marker miRNA or miRNAs, or miRNA gene product or products. The methods of treatment and pharmaceutical compositions relate to down-regulation or up-regulation of a miR gene product or products, or isolated miR gene products or miR expression inhibitor compounds respectively. OBJECT AND SUMMARY OF THE INVENTION

One object of the present invention is to provide use of a nucleic acid or acids for the manufacture of a medicine for the treatment of breast cancer.

Another object of the present invention is to provide a method of obtaining a marker value or values useful for differentiating patients with an oestrogen dependent breast cancer with a increased risk of progression into an oestrogen independent breast cancer.

A further object of the present invention is to provide a method for the treatment of oestrogen dependent breast cancer and/or prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer.

The present invention provides a use of a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any one of said nucleic acids defined in a) or b) comprising a chemical modification or modifications and able to modulate the expression of ERa; c) any nucleic acid having at least 85 % identity to said nucleic acids as defined in a) or b) and able to modulate the expression of ERa; and d) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said nucleic acids defined in a), b) or c); e) any precursor, i.e. any nucleic acid that can be processed through natural processing or synthetic processing into any of said nucleic acids as defined in a), b), c) or d); single-stranded or double-stranded, and any combination thereof for the manufacture of a medicine for the treatment of breast cancer.

The present invention also provides a method of obtaining a marker value or values useful for differentiating patients with an oestrogen dependent breast cancer with an increased risk of progression into an oestrogen independent breast cancer. Characteristic for the method is that a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any precursor, i.e. any nucleic acid that can be processed through natural processing into any of the nucleic acids defined in a); single-stranded or double-stranded, and any combination thereof are determined from a sample or samples of a tumour of the breast cancer and a marker value and/or values, which are a determined amount or amounts as such, or a function of the determined amount or amounts, are established.

The present invention further provides a method i) for the treatment of oestrogen dependent breast cancer, and/or ii) prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer in human wherein treatment comprises introducing into a tumour of said breast cancer a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any one of said nucleic acids defined in a) or b) comprising a chemical modification and able to modulate the expression of ERa; c) any nucleic acid having at least 85 % identity to nucleic acids as defined in a) or b) and able to modulate the expression of ERa; and d) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said nucleic acids defined in a), b) or c); e) any precursor, i.e. any nucleic acid that can be processed in vivo through natural processing or synthetic processing into any of the nucleic acids defined in a), b), c) or d); single-stranded or double-stranded, and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates an lysate microarray LMA screen for identifying mi RNAs regulating ERa.

Figure 2A and 2B illustrate validation of ERa regulating miRNAs.

Figure 3 illustrates that miRNA-mediated downregulation of ERa suppresses oestrogen-stimulated growth of MCF-7 breast cancer cells.

Figure 4 shows that miR-18a, miR-18b, miR-193b, and miR-206 induce accumulation of MCF-7 cells in G₁ZG₀ phase of the cell cycle.

Figure 5A and 5B show that ERa is a direct target for miR-18a/b, miR-193b, miR- 206, and miR-302c.

Figure 6 demonstrates that expression of miR-18a and miR-18b shows inverse correlation with ERa

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to that the inventors have identified twenty or so miRNAs from hundreds of known miRNAs that clearly affect the level of ERa and its mRNA in breast cancer cells. Thus, a level of these miRNAs in a cell which deviates from that of normal cells suggests a deviating amount of ERa protein in the cell. By determining the level of these miRNAs in a tumour cell one can asses which particular miRNA-molecules can be involved in development and growth of this particular tumour and accordingly also predict whether this miRNA could be used for treatment of this particular tumour.

Definitions

As used herein a functional fragment of a nucleic acid is a fragment of a nucleic acid sequence able to modulate the expression of ERa. As used herein, an miRNA molecule is a small RNA molecule, typically 21-23 nucleotides, encoded by the human genome or produced synthetically with a sequence which corresponds to one encoded by the human genome. miRNA molecules may be single-stranded or double- stranded, and contain chemical modifications, such as the substitution of phosphate bonds with phosphorothioate bonds, the methylation of the oxygen at position 2 in the ribose (2'-O-methyl), the addition of an extra bridge between the carbons of the ribose ring (locked nucleic acid or LNA), the substitution of the sugar backbone by a pseudopeptide backbone (peptide nucleic acid, i.e. PNA) or a morpholino ring (morpholino) and the attachment of a cholesterol at the 3' OH end [Bumcrot et al. Nat Chem Biol. 2006, 12(2)71 1 -719; Castanotto and Rossi, Nature 2009, 457: 426-433]. It should be emphasized that the modifications mentioned above are only non-limiting examples.

miRNA refers to the unprocessed (precursor) or processed (mature) RNA transcript from a miR gene. The unprocessed miR gene transcript is also called a miR precursor or pre- miR and typically comprises an RNA transcript of 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNAse into an active typically 21-23 nucleotide RNA molecule. This active typically 21-23 nucleotide RNA molecule is also called the processed miR gene transcript or mature miRNA. The mature miRNA molecule can be obtained from the miR precursor through natural processing or by synthetic processing. The mature miRNA molecule can also be produced directly by biological or chemical synthesis, without having been processed from the miR precursor.

miRNAs disclosed in table 2 contain a seed sequence of 6-7 nt in the 5'-end (nucleotides at position 2-8, disclosed in table 3) displaying Watson-Crick base pairing with the target mRNA [Lewis et al. Cell 2003, 1 15(7): 787-798, Friedman et al. Genome Res. 2009 19 (1 ):92-105]. Additional determinants for target binding by miRNA include non-Watson- Crick recognition of an A at nucleotide position 1 and of an A or U at nucleotide position 9 [tables 2 and 3; Lewis et al. Cell 2005, 120 (1 ):15-20; Nielsen et al. RNA 2007 13(1 1 ): 1894-1910].

When a microRNA (miR) is referred to herein by name, the name corresponds to both the precursor and mature forms, unless otherwise indicated. Table 1 and 2 depict the nucleotide sequences of particular precursor and mature human microRNAs, respectively. The seed sequence determinants of each miRNA are disclosed in table 3. In addition to the full-length miRNA molecules, such as those shown in sequences SEQ ID NOS: 1-42, the term miRNA molecules also include fragments of miRNA molecules provided that the fragments are functional fragments. The term fragment of a miRNA molecule means a portion of the full-length molecule. The size of the fragment is limited only in that it must be a functional fragment, that is, able to modulate the expression of ERa.

Naming of miRNAs both precursor (see e.g. table 1 ), mature (see e.g. table 2) or miRNA seed sequences refer to sequences provided at Sanger Center miRBase with accession numbers as disclosed in tables 1 to 3.

By modification is meant any biochemical or other synthetic alteration of a nucleotide, amino acid, or other agent relative to a naturally occurring reference agent.

Preferred embodiments of the invention

The present invention involves applications of known miRNA sequences and sequences related to these known miRNA sequences. Known precursors to the miRNA sequences related to the invention are listed in table 1. The miRNA sequences are listed in table 2 and the seed sequences of these miRNA sequences are listed in table 3.

Table 1 Precursor miRNA (pre-miR) sequences

A typical use according to the invention of the sequences involved in the invention is use of a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said SEQ ID NOS: 24-42; c) any one of said nucleic acids defined in a) or b) comprising a chemical modification or modifications; d) any nucleic acid having at least 85 % identity to said nucleic acids as defined in a), b) or c); and e) any precursor, i.e. any nucleic acid that can be processed through natural processing or synthetic processing into any of said nucleic acids as defined in a), b), c) or d); single-stranded or double-stranded, and any combination thereof for the manufacture of a medicine for the treatment of breast cancer.

In preferred embodiments the nucleic acid is RNA or a derivative thereof, i.e. RNA comprising a chemical modification or modifications. Typically the chemical modification or modifications are selected from the group consisting of substitution of a phosphate bond with a phosphorothioate bond, methylation of the oxygen at position 2 in ribose, addition of an extra bridge between the carbons of the ribose ring, substitution of the sugar backbone by a pseudopeptide backbone or a morpholino ring, attachment of a cholesterol at the 3^'OH end, and any combination thereof.

Typical preferred embodiments involve constructs which either comprise mature miRNAs or result in the target cell, through processing, in mature miRNAs, e.g. as defined in a), c) or d) above.

In many preferred embodiments the use of the nucleic acids referred to in a) of the use above are selected from the group consisting of SEQ ID NOS: 24-28 and 31- 34, and preferably from the group consisting of SEQ ID NOS: 24-27.

If functional fragment or fragments as referred to in b) above are used said functional fragment or fragments preferably consists of 7 to 22, or 12 to 22, and most preferably of 12 to 17 nucleotides; each including a seed sequence, i.e. any one of SEQ ID NOS: 43 to 54.

If a precursor or precursors as referred to in e) above are used said precursor typically consists of 25 to 200, preferably 50 to 150, and most preferably 80 to 100 nucleotides. In some preferred embodiments the precursor used is selected from the group consisting of any one of SEQ ID NOS: 1-23.

In preferred embodiments of the invention the medicine is manufactured for the treatment of a) oestrogen dependent breast cancer and/or b) the prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer.

In many preferred embodiments the use of the nucleic acid or acids as defined above is combined with the use of an antioestrogen. Preferably the antioestrogen is selected from the group consisting of fulvestran, letrozole, raloxifene, tamoxifen and toremifene.

A typical method of the invention involves obtaining a marker value or values useful for differentiating patients with an oestrogen dependent breast cancer with an increased risk of progression into an oestrogen independent breast cancer wherein nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any precursor, i.e. any nucleic acid that can be processed through natural processing into any of the nucleic acids defined in a) or b); single-stranded or double-stranded, and any combination thereof are determined from a sample or samples of a tumour of the breast cancer and a marker value and/or values, which are a determined amount or amounts as such, or a function of the determined amount or amounts, are established. Depending on the marker or markers determined; and/or if the determined amount or amounts are used as a marker or markers as such, or if a function of the determined amount or amounts are used as a marker or markers; an increased or decreased marker value suggests an increased risk of progression of an oestrogen dependent breast cancer into an oestrogen independent breast cancer. E.g. increased miR-18a and/or miR18b levels suggest an increased risk of progression of an oestrogen dependent breast cancer into an oestrogen independent breast cancer.

Preferably the nucleic acid or acids are determined by any target polynucleotide recognizing detection method, preferably a method employing nucleic acid amplification. Another method of the invention involves i) treatment of oestrogen dependent breast cancer, and/or ii) prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer in human wherein treatment comprises introducing into a tumour of said breast cancer a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said SEQ ID NOS: 24-42; c) any one of said nucleic acids defined in a) or b) comprising a chemical modification; d) any nucleic acid having at least 85 % identity to nucleic acids as defined in a), b) or e); and e) any precursor, i.e. any nucleic acid that can be processed through natural processing or synthetic processing into any of the nucleic acids defined in a), b) or e); single-stranded or double-stranded, and any combination thereof.

The invention also involves a method for downregulating oestrogen receptor ERa expression levels in breast cancer cells comprising administering miRNAs as defined above to said breast cancer cells.

The invention further involves a method for inhibiting oestrogen dependent cell proliferation, comprising administering miRNAs to oestrogen receptor containing cells in an amount effective to inhibit proliferation in said cells.

The typical and preferred embodiments disclosed above relate to all aspects of the invention disclosed in this application.

miRNA precursors or miRNA mimics can be chemically synthesized or recombinantly produced. There are many commercial suppliers for synthetic miRNA mimics which are designed to enter the miRNA pathway and act as mature miRNA species. The commercial suppliers include e.g. Dharmacon Inc. (Chicago, IL), Ambion Inc. (Austin, TX), Exiqon (Vedbaek, Denmark), and Qiagen Inc. (Valencia, CA). miRNA precursors can also be expressed from DNA plasmids containing miRNA precursor sequences using suitable promoters, such as Pol II, Pol III or the cytomegalovirus (CMV) promoters.

Normal, unmodified RNA has low stability under physiological conditions because of its degradation by ribonuclease enzymes present in the living cell. If the oligonucleotide shall be administered exogenously, it is highly desirable to modify the molecule according to known methods to enhance its stability against chemical and enzymatic degradation. Modifications of nucleotides to be administered exogenously in vivo are extensively described in the art [Bumcrot et al. 2006] [Castanotto and Rossi, 2009]. The affinity, average half-life, toxicity and/or delivery of miRNAs can be improved through chemical modifications, such as the substitution of phosphate bonds with phosphorothioate bonds, the methylation of the oxygen at position 2 in the ribose (2'-O-methyl), the addition of an extra bridge between the carbons of the ribose ring (locked nucleic acid or LNA), the substitution of the sugar backbone by a pseudopeptide backbone (peptide nucleic acid or PNA) or a morpholino ring (morpholino) and the attachment of a cholesterol at the 3' OH end. It should be emphasized that the modifications mentioned above are only non-limiting examples.

A number of strategies for the delivery of miRNA precursors or plasmids encoding the miRNA precursors could be applied to the miRNA-based therapy. Successful administration of siRNAs has already been reported in the livers of mice and non- human primates using chemically modified duplexes with cholesterol conjugation or liposomal formulation [Soutschek et al. Nature 2004; 432:173-8, Zimmermann et al. Nature 2006; 441 :1 1 1 -4, Morrissey et al. Nat Biotechnol 2005; 23:1002-7], and these strategies could be applied for miRNA delivery as well. The activity of therapeutic miRNAs can be enhanced by chemical modifications and by an adjuvant that protects it from degradation and makes sure that the therapeutic miRNA reaches the diseased tissue. Chemically modified miRNA mimics can be delivered to the cells by using suitable lipid-based transfection reagents or cholesterol-conjugates, which are well known in the art, and have been succesfully used in miRNA in vivo studies [Krϋtzfeldt et al. Nature 2005, 685-689] [Elmen et al. Nature 2008, 452: 896-899]. In addition, ligands for specific cell surface receptors capable of being internalized can be conjugated to the miRNA-oligonucleotides, thereby facilitating both cellular uptake and cell type-specific delivery [Juliano et al. Nucleic Acids Res 2008, 36:4158-71 ]. The use of supramolecular nanocarriers, such as liposomes, and polymeric nanoparticles represents another potential strategy for delivering miRNAs [Juliano et al. Nucleic Acids Res 2008, 36:4158- 71 ]. In addition, a prostate cancer xenograft model has revealed that atelocollagen can efficiently deliver synthetic miRNAs to tumor cells on bone tissues in mice when injected into tail veins [Takeshita et al. MoI Ther 2009, 18(1 ):181 -7].

An alternative means of triggering miRNA overexpression is through promoter- driven miRNA expression cassettes encoding miRNA precursors, which are processed as mature, active miRNAs in the target cells. These expression cassettes can be inserted into the backbones of viral vectors under the control of Pol II, Pol III, or CMV promoters. A potential advantage of vector delivery is that a single administration triggers long-term expression of the therapeutic miRNA. Any viral vector which is capable for delivering miRNA precursors can be used. These include, but are not limited to, e.g. lentiviruses, adenoviruses, or adeno-associated viruses. Several proof of concept studies have been carried out, with one example showing that overexpression of let-7 by an adenovirus can reduce hyperplasia in a mouse model of lung cancer [Esquela-Kerscher et al. Cell Cycle 2008, 7:759-64]. Other examples of successful delivery of miRNAs via viral vectors are provided in the literature [McLaughlin et al. PNAS 2007, 104(51 ): 20501 -20506] [Gentner et al. Nature Methods 2009, 6(1 ):63-66].

EXPERIMENTAL PART

We applied the protein lysate microarray (LMA) technology for rapid systematic profiling of miRNAs that directly impact ERa levels in breast cancer cells. We transiently transfected a library of 319 pre-miRNAs in MCF-7 and BT-474 cells and studied their impact on ERa protein expression. We identified 21 novel miRNAs which potently modulated ERa protein and mRNA levels in MCF-7 cells, and showed that miR-18a/b, miR-193b, miR-206, and miR-302c directly targeted ERa by binding to its 3'UTR region and inhibited ERa target genes. Furthermore, miR- 18a and miR-18b levels were significantly higher in vivo in ERα-negative breast cancer, suggesting a role for these miRNAs in the development of ERa negative breast cancers.

Materials and methods

Cell Culture and Reagents MCF-7 cells were obtained from lnterlab Cell Line Collection (ICLC, Italy) and BT- 474 from American Type Culture Collection (ATCC). MCF-7 cells were cultured in DMEM (1 g/l glucose) supplemented with 10 % foetal bovine serum (FBS), 2 mM L-glutamine and 1 % penicillin/streptomycin. BT-474 cells were cultured in DMEM (4.5 g/l glucose) supplemented with 10 % FBS, 4 mM L-glutamine, 0.01 mg/ml insulin, 1 mM Na-pyruvate, and 1 % penicillin/streptomycin.

Human Pre-miR™ miRNA Precursors and an siRNA for human oestrogen receptor-α (GGCCAAAUUCAGAUAAUCGTT; SEQ ID NO:55) were purchased from Ambion (Austin, TX), and used at a final concentration of 20 nM. β-estradiol (oestrogen) and 4'OH-Tamoxifen were purchased form Sigma-Aldrich (St. Louis, MO).

Lvsate Microarrav Screening and Data Analysis

MCF-7 and BT-474 cells were transfected with 20 nM human Pre-miR™ miRNA Precursor library v2 (Ambion Inc., Austin, TX) containing 319 chemically modified double-stranded RNA molecules designed to mimic endogenous mature human miRNAs. Briefly, the pre-miRNAs were printed robotically to 384-well black, clear- bottom assay plates (Greiner Bio-One GmbH, Frickenhausen, Germany). SilentFect™ transfection agent (BioRad Laboratories, CA) diluted into OptiMEM (Gibco Invitrogen, CA) was aliquoted into each 384-plate well using Multidrop 384 Microplate Dispenser (Thermo Labsystems, Thermo Electron Corporation, MA), and the plates were incubated for 1 h at room temperature. Subsequently, 35 μl of cell suspension (1500 MCF-7 or 2000 BT-474 cells) was added on top of the miRNA-lipid complexes and the plates incubated for 48 h or 72 h.

After transfections, the cells were lysed by adding 15 μl of lysis buffer (100 mM Tris, pH 8.0; 0.2% SDS; 25 mM DTT) directly onto the 384-well plates. Thereafter, the lysates were denatured in 95 °C for 15 min, and stored at -20 °C prior printing. Microarray printer QArray² with 32 split pins (Genetix) was used to print arrays on nitrocellulose-coated microarray slides (FAST slides, Whatman inc.). Four drops of the cell lysates were stamped to each spot position ending up with a total of 3072 spots on a slide (-300 μm in diameter). Each slide was blocked with near- infrared blocking buffer (Rockland Immunochemicals) for 1 h at room temperature. PAP pen was used to draw a hydrophobic barrier around the nitrocellulose membrane to keep 500 μl of the sample volume stably on the membrane during incubations. ERa antibody (Ab-15; Labvision Corp, Fremont, CA) incubation at dilution of 1 :200 was done overnight at 4 °C in a humidified chamber to prevent the slides from drying. The slides were washed with TBS + 0.1 % Tween-20 for three times 5 min, and exposed to Alexa Fluor 680 tagged secondary antibody (Invitrogen Inc.) at a dilution of 1 :5000 for 45 min. For total protein measurement, the arrays were stained with Sypro Ruby Blot solution (Invitrogen Inc.). Sypro signal was detected using Tecan LS400 (Tecan inc.) microarray scanner at 488/670 nm, and Alexa Fluor 680 label at 700 nm using IR-scanner (Li-Cor Inc.). Array-Pro Analyzer Microarray Analysis Software (Median Cybernetics Inc.) was used to measure median pixel intensities of each spot and the slide background from each channel. After background subtraction, spot intensity was normalized to the median intensity of pin to correct pin-to-pin as well as spatial variation on the array. Net signal from antibody staining was acquired after normalization to the Sypro signal. Finally, normalized values were converted to Z-scores. Candidate miRNAs down-regulating ERa were picked for secondary assays from Z-score window determined by positive and negative control siRNAs (ESR1 siRNA, Qiagen) and miRNAs (Scrambled, Ambion) respectively. Two target prediction methods were used to assess the specificity of ERa downregulation after miRNA transfection. The TargetScanS v.4.2 [Grimson A et al. MoI Cell 2007; 27(1 ):91 -105] and mirDB v.2.0 [Wang X and El Naqa IM Bioinformatics 2008; 24(3):325-32] were used to obtain miRNA target predictions. Enrichment factor and p-value according to the hypergeometric distribution was calculated using the minimum Z-score value of the negative control scrambled miRNAs as a threshold. Additionally, a t-test was used to show that the miRNAs predicted to target ERa were more effective in reducing the ERa levels than the miRNAs not predicted to target ERa.

Immunoblottinq

Aliquots of total cell lysates were fractionated on SDS-polyacrylamide gels and transferred to Whatman Protran nitrocellulose membrane (Schleicher & Schuell, Whatman Inc, Florham Park, NJ). The filters were blocked against non-specific binding using 5 % skim milk. Membranes were probed with a specific antibody for human ERa (Ab-15; Labvision Corp, Fremont, CA). Equal loading was confirmed by probing the same filter with a specific antibody for human β-actin (Sigma, St. Louis, MO). Signals were revealed by incubating the filters with secondary antibody Alexa Fluor 680 anti-mouse IgG (Invitrogen Corp, Carlsbad, CA) and scanning the filters with Odyssey Licor (LI-COR Biosciences, Lincoln, NE). The relative levels of ERa protein were quantitated with Odyssey LI-COR 2.1 software and normalized for β-actin.

Real-time quantitative PCR analysis

Total cellular RNAs were isolated with MiRVana™ Total RNA isolation kit (Ambion Inc., Austin, TX). For cDNA synthesis, 200 ng of total RNA was reverse transcribed with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Thereafter, the cDNAs were diluted 1/10 and the Taqman quantitative real-time PCR (qRT-PCR) analysis performed with Applied Biosystems 7900HT instrument using specific primers for ERa (ESR1 ) and β-actin (ACTB) designed by the Universal Probe Library Assay Design Center (Roche Applied Biosciences, Basel, Switzerland). The sequences of the primers were as follows: ESR1 forward δ'-TTACTGACCAACCTGGCAGA-S' (SEQ ID NO:56), ESR1 reverse δ'-ATCATGGAGGGTCAAATCCA-S' (SEQ ID NO:57), ACTB forward 5'-CCAACCGCGAGAAGATGA-S' (SEQ ID NO:58), ACTB reverse 5'-CCAGAGGCGTACAGGGATAG-S' (SEQ ID NO:59). The fluorescent Taqman probes were obtained from Roche Human Probe Library (#24 for ESR1 and #64 for ACTB). The results were analyzed with SDS 2.3 and RQ manager software (Applied Biosystems, Foster City, CA), and the expression of ESR1 mRNA determined by relative quantitation method using β-actin as an endogenous control. The data is from two separate biological experiments, which were both run twice with triplicate samples.

ER 3'UTR reporter constructs and luciferase assays

Four separate fragments covering the ERa 3'-UTR region were amplified from the genomic DNA of MCF-7 cells using the following primers: ER#1_13-961 , sense 5'-ATATACTAGTCCCACACGGTTCAGATAATC-S' (SEQ ID NO:60) and antisense 5'-ATATACGCGTACCCTCATCATGCATCACCA-S' (SEQ ID NO:61 ); ER#2_91 1 -2050, sense 5'-ATATACTAGTGCCTTACACAGGGGTGAACT-S' (SEQ ID NO:62) and antisense 5'-ATATACGCGTTTTGCATGTTAACCCAGTCA-S' (SEQ ID NO:63); ER#3_2032-3222, sense δ'-ATATACTAGTCTGGGTTAACAT- GCAAAAACC-3' (SEQ ID NO:64) and antisense δ'-ATATACGCGTTTGTTCAG- CCCTCAAAAGG-3' (SEQ ID NO:65); E R#4_3201 -4243, sense 5'-ATATACTAG- TCCTTTTGAG G G CTG AACAAA-3' (SEQ ID NO:66) and antisense 5'-ATATAC- GCGTCCCGCTGGATTCTTTTTCAA-3' (SEQ ID NO:67). The resulting ERa 3'UTR fragments were cloned into the Spe/Mlul sites of a pMIR-REPORT™ Luciferase vector (Ambion Inc, Austin, TX) downstream of a luciferase gene.

For luciferase assays, MCF-7 cells (15000 per well) were plated 24 h before transfection onto white, clear-bottom 96-well plates in normal culture medium without antibiotics. The cells were co-transfected with 50 ng of ER 3'UTR reported plasmid, 50 ng Renilla luciferase plasmid, and with 50 nM pre-miR construct with Lipofectamine 2000 (Invitrogen Corp, Carlsbad, CA) according to the manufacturer's protocol. The luciferase activity was assayed 24 h after transfection with Dual-Glo Luciferase Assay System (Promega Corp, Madison, Wl) and measured with Envision Plate-reader (Perkin Elmer Inc, Wellesley, MA).

Proliferation assays

In experiments for assaying the effect of miRNAs on the oestrogen-stimulated growth of MCF-7 cells, the cells were cultured in phenol-red-free DMEM (Sigma,

St. Louis, MO) supplemented with 10 % charcoal-stripped FBS and 2 mM

L-glutamine. The cells (10000 per well) growing on white, clear-bottom 96-well plates were transfected with 20 nM pre-miR constructs (Ambion) or with siERα using SiLentFect (Bio-Rad). After 24 h incubation, the cells were treated with 2 nM 17β-estradiol or DMSO (vehicle), as indicated, for 72 h. Cell proliferation was assayed with CellTiter-Glo cell proliferation assay (Promega Corp, Madison, Wl).

Cell cycle analysis

The cell cycle analyses were done from the nuclei preparations of MCF-7 cells. Cells (30000 per well on 24-well plates) were transfected with 20 nM pre-miR constructs or with siERα and incubated for 48 h. Thereafter, the cells were trypsinized, processed for the cell cycle analysis with BD CycleTest Plus DNA reagent kit (BD Biosciences, San Jose, CA), and examined for the cell cycle distribution with FACSArray (BD Biosciences, San Jose, CA). The data was analyzed using Multicycle software (Phoenix FlowSystems, San Diego, CA).

lllumina gene expression analyses

MCF-7 cells (300 000 per well on 6-well plates) were transfected with an si RNA for ERa or with Ambion pre-miR™ constructs for miR-18a, miR-193b, miR-206, miR- 302c, or pre-miR negative control #1 (scrambled pre-miR) at 20 nM, and incubated for 24h. Thereafter, the total cellular RNAs were isolated with MiRVana™ total RNA isolation kit (Ambion). The quality of the RNAs was assayed with Agilent 2100 Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA). For lllumina gene expression profiling, 400 ng of total RNA was amplified and labelled with lllumina Total Prep RNA Amplification kit (Ambion) and hybridized on to lllumina Sentrix Human-6 Expression BeadChips. Statistical analysis of differential gene expression after mi RNA transfection was performed with R/Bioconductor [Gentleman RC et al. Genome Biol 2004; 5(10):R80] using the limma [Smyth GK Stat Appl Genet MoI Biol 2004; 3:Article3] and lumi [Du P, Kibbe WA and Lin SM Bioinformatics 2008; 24(13) :1547-8] packages. Gene expression after siRNA or miRNA transfections was compared to the scrambled miRNA negative control in pairwise fashion using the eBayes statistic. The threshold for differential expression was q<0.05 after the Benjamini-Hochberg multiple testing correction. In order to display the differentially expressed genes, hierarchical clustering was performed with R after dividing the data into similar groups using the package "cluster" and the partitioning around medoids (PAM) method [Kaufman L and Rousseeuw PJ. Finding groups in data: An introduction to cluster analysis. New York: J. Wiley and Sons, Inc.; 1990]. The Gene Set Analysis (GSA) R package [Efron B and Tibshirani R Ann Appl Stat 2007; 1 (1 ):107-29] was used for defining enriched gene sets in the data. Gene sets were obtained from the Molecular Signatures Database (MSigDB, Broad Institute of MIT and Harvard, Cambridge, MA, USA) [Subramanian A et al. Proc Natl Acad Sci U S A 2005; 102(43) :15545- 50]. The MSigDB curated gene sets (C2, 1892 sets) and motif gene sets (C3, 837 sets) including the 3'-UTR miRNA binding motifs (222 sets) [Xie X et al. Nature 2005; 434(7031 ):338-45] were downloaded from the GSEA web site. A false discovery rate (FDR) cut-off of 0 was used in all analyses.

MicroRNA expression and immunohistochemistry analyses of breast cancer samples

RNA from 96 breast cancer samples was extracted as described before [Sørlie T et al. BMC Genomics 2006; 7:127]. The samples were hybridized in two replicates (except four samples that were run once) on Human miRNA Microarray (v2) from Agilent Technologies according to the manufacturer's protocol (G4170-90010 v1.5). We used the Agilent Microarray Scanner (G2565A) and scanned at 100 % and 5 % PMT. Feature Extraction version 9.5.3.1. was used to extract signals intensites and filter the detectable miRNAs. Log2 values were used. The tumours where graded by immunohistochemistry staining of ERa, depending on the percentage of cells nuclei that were stained [Naume B et al. MoI Oncol 2007; 1 (2):160-71 ]. Twenty-eight tumours were ERa negative, and 68 tumours with staining over 1 % were considered as ERa positive. Of these, 8 tumours had 1- 10 %, 9 tumours had 10-50 %, 1 1 tumours 50-75 %, and 40 tumours have 75- 100 % cells with ESR1 positive staining. T-test and non-parametric Kruskal Wallis test were performed for association between mi RNA expression level and ER status/grading in SPSS 15.0 (SPSS Inc., Chicago, IL, USA).

Results

Lvsate microarrav screens revealed several putative ERg regulating miRNAs

Protein lysate microarray (LMA) technology, or reverse-phase protein microarray, allows proteomic profiling of a large number of biological samples with specific antibodies. We applied LMA technology in order to identify ERg targeting miRNAs in breast cancer cell lines. MCF-7 and BT-474 cells were transfected with Pre- miR™ miRNA library containing 319 chemically modified, double-stranded pre- miR constructs mimicking endogenous miRNAs. After 48 h and 72 h incubation, the cells were lysed and the lysates printed on nitrocellulose slides to be stained with a specific antibody against ERg. The slides were also counterstained with Sypro Ruby in order to normalize the levels of ERg for the total protein. With LMA screening, we identified 60 and 93 miRNAs which reduced the expression of ERg in both cell lines at 48 h and 72 h time points, respectively (Figure 1 ). The siRNA for ERg (siERg) was used as a positive control. Among the miRNAs that downregulated ERg was miR-206, which has previously been shown to directly target ERg [Adams BD et al. 2007] (Figure 1 ). Furthermore, miRNAs predicted to target ERg according to various prediction algorithms were enriched among the ERg downregulating miRNAs. Two sample Wilcoxon test p-values for the enrichment were highly significant for MCF-7 cells at 48 h and 72 h timepoints (p< 2.1 e^"06 and p<1.2e^"06, respectively) . The p-values for BT-474 were also <0.05 at both time points (not shown).

Figure 1, LMA screen for identifying miRNAs regulating ERa

MCF-7 and BT-474 breast cancer cells were transfected with Pre-miR miRNA precursor library containing 319 pre-miR constructs and incubated for 48 h or 72 h. Thereafter, the cells were lysed, printed onto nitrocellulose coated slides, and stained with a specific antibody for ERa. Slides were counterstained with Sypro Ruby in order to normalize the total protein amount. Figure 1 shows representative LMA screens of MCF-7 and BT-474 cells plotted against each other.

Validation of the Ivsate microarrav data

To validate the primary LMA screening data, we selected miRNAs which were identified in at least two replicate LMA screens as putative ERa downregulating miRNAs. As an additional selection criteria we used miRNA target predictions from TargetScan [Grimson A et al. (2007)], PicTar [Krek A et al. Nat Genet 2005; 37(5):495-500], and MiRanda [Betel D et al. Nucleic Acids Res 2008; 36(Database issue):D149-53]. We were then left with 21 miRNAs indicated in Table 4. In western blotting, all these miRNAs downregulated ERa protein levels in MCF-7 and BT-474 cells by up to 80 %, validating the LMA primary screening data (Figure 2A). Transfections of pre-miR constructs for miR-200a and miR-200b, not predicted to target ERa and not scoring positive in the LMA screens, served as negative controls (Figure 2A).

In addition to translational repression, miRNAs regulate the gene expression by targeting mRNAs for cleavage. We therefore examined the effect of pre-miR overexpression on the levels of ERa mRNA with real-time qRT-PCR. Transfection of MCF-7 cells with siERα reduced the levels of ERa mRNA by 2-fold (Figure 2B). The miRNAs miR-18a/b, miR-22, miR-93, miR-130a/b, miR-302c, miR-372, miR- 373, and miR-520d also downregulated ERa mRNA levels, suggesting that these miRNAs target ERa mRNA to degradation (Figure 2B). In comparison, miR-181d, miR-193b and miR-206 had only minor effects on ERa mRNA levels, indicating that these miRNAs may regulate ERa mainly at the translational level. Negative control miRNAs, miR-200a/b, did not significantly affect the levels of ERa mRNA in MCF-7 cells (Figure 2B). Tabfe 4. LIVlA Z -scores and ERa predictions for the miRNAs selected for validation.

The data is from MCF-7 screens performed at 48 h and 72 h time points (sorted according to 72 h).

ERα/sypro z-score Predicted sites in the ERa 3'UTR

TargetScan, TargetScan,

Sample 48h 72h PicTar* MiRanda^s conserved non-conserved

ERa siRNA -1.27 -2.75 hsa-miR-206 -0.94 -2.74 7mer~m8, 7mer-1A 1 hsa-miR-219 -2.43 -2.54 7mer-m8 1 1 hsa-miR-222 -2 78 -2 40 8mer 7mer-1A 1 1 hsa-miR-9 -1.70 -2.16 7mer-1A 7mer-fτi8 2 1 hsa-miR-51 ?a -1 68 -1.82 1 hsa-rniR-18a -0.47 -1.77 δrner 8mer, 7fner-1A 3 2 hsa-miR-373 -0.64 -1.69 7mer-m8 7mer-1A 2 hsa-miR-181 d -1.16 -1.64 8mer 2 hsa-miR-302c -1.68 -1.57 7mer-m8 7mer-1A 2 1 cn hsa-miR-301 -0.87 -1.56 7mer-m8 7mer-fτi8, 7mer-1A 2 2 hsa-miR-372 -1.69 -1.53 7mer-m8 7mer-1A 2 hsa-miR-193b -049 -1 42 1 hsa-miR-13Ga -0.96 -1.41 7mer-m8 7mer-m8. 7mer-1A 2 2 hsa-miR~130b -0.71 -1.27 7mer-m8 7mer~m8. 7mer-1A 2 1 hsa~miR-181 a -1.32 -1.19 Srner 1 1 hsa-miR-517c -0.80 -1.14 1 hsa-miR-1δb -1.13 -1.08 8mer 8mer, 7mer-1A 3 2 hsa-miR-22 -1.18 -0.89 Smer 7mer-m8. 7mer-1A 3 1 hsa-miR-93 -0.37 -0.84 7mer-m8 7mer-1A 2 2 hsa-miR-520d -1.18 -0.25 7mer-m8 7mer-1A hsa-miR-181 c -0 95 -0.08 8mer 1 1 pre-mir neg.ctrl 0.39 0.06 cells only Ctrl -Q 10 0 10

""For PicTar and MiRanda the number of predicted sites is shown.

Figure 2A, Validation of ERa regulating miRNAs

MCF-7 and BT-474 breast cancer cells were transfected with pre-miR constructs (20 nM) and incubated for 48 h. Thereafter, the cells were lysed and the lysates analyzed with western blotting for the expression of ERa.

Figure 2B, Validation of ERa regulating miRNAs

MCF-7 breast cancer cells were transfected with pre-miR constructs and incubated for 24 h. Thereafter, the total cellular RNAs were isolated and analyzed with Taqman qRT-PCR for the expression of ERa mRNA. The results normalized for β-actin are shown as relative expression using scrambled negative control miRNA (Scr) as a reference. Mean ± SEM (n=4).

MiRNA-mediated down regulation of ERa suppresses oestrogen-stimulated growth of MCF-7 cells

Oestrogen stimulates the proliferation of hormone-dependent breast cancer cells, and this is mediated primarily via ERa [Couse JF and Korach (1999)]. To study the functional significance of the ERα-downregulating miRNAs, we analyzed whether the miRNA-mediated downregulation of ERa could inhibit the growth stimulatory effects of oestrogen on MCF-7 cell growth. For that purpose, the MCF-7 cells were first cultured in hormone-free growth media for 24 h, and then transfected with the corresponding pre-miR constructs. After additional 24 h incubation, the cells were treated with oestrogen 17β-estradiol (2 nM) or with vehicle (DMSO) control for 72 h. Thereafter, the proliferation was assayed, and the fold-induction between oestrogen and vehicle treated samples measured. Oestrogen stimulation enhanced the growth of negative control pre-miR transfected MCF-7 cells by 2.4-fold, whereas pre-treatment of cells with 4'OH-Tamoxifen (OHT) totally suppressed the oestrogen-stimulated growth of MCF-7 cells (Figure 3). When ERa was knocked down with siERα, the oestrogen-induced growth was inhibited by 60 % (Figure 3). Most of the miRNAs showed similar or slightly lower inhibition as siERα. The most potent growth inhibitory miRNAs were miR-93, miR-193b, miR- 222, miR-302c, miR-372, and miR-520d (Figure 3). In comparison, transfection of cells with an siRNA for ERβ had no significant effect confirming that growth inhibition was specifically dependent on ERa.

Figure 3, miRNA-mediated down regulation of ERa suppresses oestrogen- stimulated growth of MCF-7 breast cancer cells

MCF-7 cells cultured in hormone-free growth media were transfected with pre-miR constructs or with an siRNA for ERa. After 24-h incubation, the cells were treated with oestrogen 17β-estradiol (2 nM) or with vehicle (DMSO) for 72 h. Cell growth was measured with CellTiter-Glo cell viability assay. Relative increase in cell number in response to oestrogen stimulation is shown in the graph. The data represent mean ± SEM from three independent experiments, each performed in triplicate (^*p< 0.05; ^***p=0.00003).

MiR-18a, miR-18b, miR-193b, and miR-206 induce accumulation of MCF-7 cells in G-i/Gn phase of the cell cycle

In the absence of oestrogens or in the presence of antioestrogens, proliferation of hormone-dependent breast cancer cells is inhibited and they accumulate in the Gi phase of the cell cycle [Doisneau-Sixou SF et al. Endocr Relat Cancer 2003;

10(2):179-86]. Therefore, we next studied how downregulation of ERa by miRNAs affects the cell cycle of MCF-7 cells. The cells were transfected with pre-miR constructs for the 21 ERa down-regulating miRNAs, and the cell cycle distribution was analyzed after 48 h by FACS. While many of the miRNAs did not change the proportion of MCF-7 cells in G₁, S, or G₂ phases of the cell cycle, miR-18a, miR-

18b, miR-193b, and miR-206 significantly induced accumulation of the cells in the

G-i/Go phase. In these experiments, the average percentage of cells in GVG₀ phase was 71 %, 68 %, 80 %, and 73 %, respectively, whereas in the controls this was 47 %. These data were again comparable with the results for siRNA-mediated

ERa knockdown as well as with antioestrogen 4'OH-Tamoxifen (OHT; 1 μM) treated samples (Figure 4). Figure 4, Mi R- 18a, mi R- 18b, mi R- 193b, and miR-206 induce accumulation of MCF -7 cells in G₁ZG₀ phase of the cell cycle

MCF-7 cells were transfected with the pre-miR constructs (20 nM) for the indicated miRNAs or with siERα, and incubated for 48 h. Thereafter, the cell cycle distribution was analyzed from the nuclei preparations with FACS. Results are shown as percentage of cells in each phase of the cell cycle. 4'OH Tamoxifen (OHT; 1 μM) was used as a positive control for G₁ arrest. Mean±SD, n=3, ^**p< 0.01.

MiR-18a, miR-193b, miR-206, and miR-302c repress ERα-responsive genes Typically, miRNAs regulate the expression of hundreds of different genes, and often target several genes on the same pathways [Lewis BP et al. Cell 2005; 120(1 ):15-20]. To better understand the role for miR-18, miR-193b, miR-206, and miR-302c in oestrogen-mediated signal transduction, we examined their effects on the gene expression profile of MCF-7 breast cancer cells by microarray. The cells were transfected with the pre-miR constructs and siERα control and incubated for 24 h, followed by analysis using lllumina Sentrix Human-6 Expression BeadChips. A clustering analysis of all the differentially expressed genes revealed genes specifically downregulated by each miRNA. Also, a consensus set of genes downregulated by all four miRNAs (miR-18a, miR-193b, miR-206, and miR-302c) and siERα included many well-known ERα-responsive genes, such as MYB, GREB1, CD44, PDZK1, IGFBP4 and STC2. When the expression profiles were compared against profiles from the Molecular Signatures Database (MSigDB) [Subramanian A et al. (2005)] using the Gene Set Analysis (GSA) software [Efron B and Tibshirani R (2007)], the most frequently enriched signature among miRNA downregulated genes was an MSigDB signature "FRASORJJP" (Table 5). This signature was originally obtained after treating MCF-7 cells with oestrogen [Frasor J et al. Cancer Res 2004; 64(4):1522-33 (35)], and it contains ERα-dependent genes. The FrasoMJP signature was highly enriched with a FDR of 0 and was ranked the first among the curated genes sets in the MSigDB (C2, 1892 sets) after both miR-18a and miR-302c treatments (Table 5). Moreover, the FrasorJJP signature was ranked in the top ten also among the miR-193b, miR-206 and siERα enriched signatures (FDR = 0). This indicates that in MCF-7 cells, and very likely in breast cancer in general, the ERa mRNA is an important target for these miRNAs.

Table 5. Expression profiles. Expression profiles obtained after overexpressing miR-18a (Table 5A), miR-193b (Table 5B), miR-206 (Table 5C), miR-302c (Table 5D), or siERα (Table 5E) in MCF-7 cells for 24 h were compared to the 1892 curated gene sets (C2) in the MSigDB v2.5. Twenty most significant signatures are displayed arranged according to descending score. Signatures present in all different microRNA transfections in bold. The most frequently enriched signature among miRNA downregulated genes was an MSigDB signature "FrasorJJP", which was originally obtained after treating MCF-7 cells with oestrogen, and which contains ERα-responsive genes.

Table 5A

Table 5B

Table 5C

Table 5D

Table 5E

ERg is a direct target for miR-18a/b. miR-193b, miR-206 and miR-302c

Based on the TargetScan predictions [Grimson A et al. (2007)], miR-18/b has the highest scoring target sites in the 3'UTR region of ERg. A conserved 8mer site can be found at position 1938-1945 of the ESR1 3' UTR with a context score percentile of 94 %. In addition, the ER 3'UTR contains one non-conserved 8-mer site and one 7mer-1 A site for miR-18a/b at positions 1917-1924 and 527-533, respectively. MiR-302c also has a favourable target site in the ERg 3'UTR with context score percentile of 86 %. MiR-193b is predicted to target ERg by another algorithm, PicTar [Krek A et al. (2005)], and it has a conserved, predicted 7mer site at position 3999-4005 of the ER 3'UTR. Since our studies suggested that miR-18a/b, miR-193b, miR-206, and miR-302c are the most potent modulators of oestrogen- receptor signalling, we analyzed whether these miRNAs directly target ERg. For that purpose, we cloned four separate fragments covering the ERg 3'UTR region into a luciferase reporter vector (Figure 5A; Table 6). The resulting ERg 3'UTR- luciferase reporter plasmids were transfected in MCF-7 cells together with the corresponding pre-miR constructs and with a Renilla luciferase control plasmid. Transfection of miR-18a, miR-18b, miR-193b, miR-206 and miR-302c significantly inhibited the reporter activity as compared to scrambled mi RNA transfected negative control, indicating that these miRNAs directly target ERg (Figure 5B). Figure 5 A, ERa is a direct target for miR-18a/b, mi R- 193b, miR-206, and miR-302c

Figure 5A illustrates a schematic representation of the subfragments of ERa 3'UTR which were inserted into the pMI R-REPORT Luciferase reporter vector. The predicted mi RNA target sites are indicated with arrows, and are listed in table 6.

Table 6. Predicted miRNA sites. miRNA Construct p_{osition jn the ERα 3}-_UTR Prediction algorithm no # hsa-miR-18a/b #1 527-533 TargetScan, PicTar #2 1917-1924 & 1938-1945 TargetScan, PicTar hsa-miR-193b #4 3999-4005 PicTar hsa-miR-206 #1 86-109 Adams et al. 2007 (RNAHybrid)¹ #2 1 152-1 158 & 1532-1538 TargetScan #4 3938-3959 MiRanda hsa-miR-302c #1 379-385 TargetScan & PicTar #2 1832-1838 TargetScan & PicTar

Predicted site in ERa 3'UTR as identified by RNAHybrid Adams et al. (2007).

Figure 5B, ERa is a direct target for miR-18a/b, mi R- 193b, miR-206, and miR-302c

Activity of luciferase reporters containing segments of the ERa 3'UTR. The reporters (100 ng) were cotransfected with pre-miR constructs (50 nM) for the indicated miRNAs, and luciferase activity measured after 24 h incubation. Firefly luciferase activity was normalized to that of Renilla luciferase. The data shown are the mean ±SEM of three independent experiments, each performed in triplicate. ^*p<0.05, ^**p<0.01

MiR-18a and miR-18b have lower expression in ERα-positive tumours

To study the clinical relevance of the miRNAs directly targeting ERa, we analyzed the miRNA microarray expression data for miR-18a, miR-18b, miR-193b, miR-206, and miR-302c in 96 primary breast cancer specimens. The analysis was done both by dichotomizing the tumors into ERα-positive and ERα-negative as well as by correlating the miRNA expression levels with the semiquantitative assessment of ERa immunostaining. The expression of miR-302c was not detected in any of the tumours, and the expression of miR-206 was detected in only 26/96 tumours. With miR-206 and miR-193b, there was no association with ERa grading (data not shown). However, the results provided a statistically significant inverse association between the expression of miR-18a and miR-18b and that of the ERa immunostaining (p=0.0001 , p=0.0004 respectively) (Figure 6). This suggests that the expression of miR-18a and miR-18b is an important in vivo regulatory mechanism of ERa in breast cancer patients.

Figure 6, The expression of miR-18a and miR-18b shows inverse correlation with ERa

Ninety-six (96) primary breast cancer specimens were analyzed for miRNA expression using Agilent miRNA microarray. The analysis was done both by dichotomizing the tumours into ERα-positive and ERα-negative as well as by correlating the miRNA expression levels with the semiquantitative assessment of ERa immunostaining. The tumours where graded (0-4) by immunohistochemistry (IHC) staining of ERa, depending on the percentage of cells nuclei that were stained (0=0 %; 1 =1-10 %; 2=10-50 %; 3=50-75 %; 4=75-100 %). The boxplots show the expression of hsa-miR-18a (left panel) and hsa-miR-18b (right panel) in relation to the ERa grading. The open dots indicate outliers.

Other preferred embodiments It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive

Claims

1. Use of a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any one of said nucleic acids defined in a) comprising a chemical modification or modifications and able to modulate the expression of ERa; c) any nucleic acid having at least 85 % identity to said nucleic acids as defined in a) or b) and able to modulate the expression of ERa; and d) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said nucleic acids defined in a), b) or c); e) any precursor, i.e. any nucleic acid that can be processed through natural processing or synthetic processing into any of said nucleic acids as defined in a), b), c) or d); single-stranded or double-stranded, and any combination thereof for the manufacture of a medicine for the treatment of breast cancer.

2. The use according to claim 1 characterized in that the nucleic acid is RNA or a derivative thereof, i.e. RNA comprising a chemical modification or modifications.

3. The use according to claim 2 characterized in that a chemical modification or modifications selected from the group consisting of substitution of a phosphate bond with a phosphorothioate bond, methylation of the oxygen at position 2 in ribose, addition of an extra bridge between the carbons of the ribose ring, substitution of the sugar backbone by a pseudopeptide backbone or a morpholino ring, attachment of a cholesterol at the 3OH end, and any combination thereof are used.

4. The use according to claim 1 , 2 or 3 characterized in that the nucleic acids referred to in a) of claim 1 are selected from the group consisting of SEQ ID NOS: 24-28 and 31-34, preferably from the group consisting of SEQ ID NOS: 24-27.

5. The use according to any of the preceding claims characterized in that a functional fragment as referred to in b) of claim 1 is used, said fragment consisting of 7 to 22, preferably 7 to 17, or 12 to 22, and most preferably 12 to 17 nucleotides including a seed sequence, i.e. any one of SEQ ID NOS: 43-54.

6. The use according to any of the preceding claims characterized in that a precursor is used consisting of 25 to 200, preferably 50 to 150, and most preferably 80 to 100 nucleotides.

7. The use according to claim 6 characterized in that the precursor used is selected from the group consisting of any one of SEQ ID NOS: 1-23.

8. The use of any of the preceding claims characterized in that the medicine is manufactured for the treatment of a) oestrogen dependent breast cancer and/or b) the prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer.

9. The use of any of the preceding claims characterized in that the use is combined with the use of an antioestrogen.

10. The use according to claim 9 characterized in that the antioestrogen is selected from the group consisting of fulvestran, letrozole, raloxifene, tamoxifen and toremifene.

11. A method of obtaining a marker value or values useful for differentiating patients with an oestrogen dependent breast cancer with an increased risk of progression into an oestrogen independent breast cancer characterized in that a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any precursor, i.e. any nucleic acid that can be processed through natural processing into any of the nucleic acids defined in a); single-stranded or double-stranded, and any combination thereof are determined from a sample or samples of a tumour of the breast cancer and a marker value and/or values, which are a determined amount or amounts as such, or a function of the determined amount or amounts, are established.

12. The method of claim 1 1 characterized in that the nucleic acid or acids are determined by any target polynucleotide recognizing detection method, preferably a method employing nucleic acid amplification.

13. A method i) for the treatment of oestrogen dependent breast cancer, and/or ii) prevention of progression of oestrogen dependent breast cancer into oestrogen independent breast cancer in human wherein treatment comprises introducing into a tumour of said breast cancer a nucleic acid or nucleic acids selected from the group consisting of a) any one of SEQ ID NOS: 24-42; b) any one of said nucleic acids defined in a) or b) comprising a chemical modification and able to modulate the expression of ERa; c) any nucleic acid having at least 85 % identity to nucleic acids as defined in a) or b) and able to modulate the expression of ERa; and d) any functional fragment, i.e. a fragment able to modulate the expression of ERa, of any one of said nucleic acids defined in a), b) or c); e) any precursor, i.e. any nucleic acid that can in vivo be processed through natural processing or synthetic processing into any of the nucleic acids defined in a), b), c) or d); single-stranded or double-stranded, and any combination thereof.