WO2011067346A1

WO2011067346A1 - Method for determining the efficiency of a cancer treatment

Info

Publication number: WO2011067346A1
Application number: PCT/EP2010/068759
Authority: WO
Inventors: Patrick Brest; Paul Hofman; Baharia Mograbi
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2009-12-04
Filing date: 2010-12-02
Publication date: 2011-06-09

Abstract

The present invention relates to an in vitro method for determining the anti-cancer activity of a compound comprising: i) measuring the level of expression of a mi-RNA selected from miR-129-5p, miR-133b and precursors thereof in cancer cells which have been in contact with said compound; ii) comparing the level of expression with a reference value; iii) determining therefrom the anti-cancer activity of the compound.

Description

Method for determining the efficiency of a cancer treatment

Field of the invention

The present invention relates to a method for determining if a compound is efficient in the treatment of a cancer, in particular if a Histone DeACetylase inhibitor (HDACi) is efficient in the treatment of cancer.

Background of the invention

Cancer is a disease caused by both genetic and epigenetic reorganizations of the chromatin structure that lead to global alteration of gene expression and play a critical role in tumour initiation and progression (Hanahan et al. (2000) Cell 100(1 ):57-70, Jones et al. (2007) Ce// 128(4):683-92).

As a central actor of epigenetic remodeling, nuclear class I histone deacetylases (HDACs) are responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation can indicate epigenetic repression by inducing local chromatin condensation and thus plays an important role in transcriptional regulation, cell cycle progression and developmental events. Interestingly, HDACs are generally over-expressed in tumors and promote tumor cell longevity by blocking the transcription of anti-tumoral genes (Bolden et al. (2006) Nat Rev Drug Discov 5(9):769-84). HDACs have thus emerged as molecular targets for the development of enzymatic inhibitors to treat human cancer. Many HDAC inhibitors (HDACi) are currently used in patients as monotherapy (Prince et al. (2009) Clin Cancer Res 15(12):3958-69) because of their activity against human malignancies with biological effects ranging from cell growth arrest, to apoptosis and induction of terminal differentiation.

However, all HDAC inhibitors do not share the same efficiency against cancer. In particular, this efficiency may vary depending on the type of tumour to be treated and also from patient to patient.

It is therefore highly desirable to implement methods enabling to evaluate the efficiency of HDAC inhibitors in order to classify patients and tumours into groups that are responsive to the drug. In various studies biomarkers have been assessed to elucidate how HDAC inhibitors exert their effect. Some sets of genes were identified in cancer cells from patient, the expression of which being indicative of response to HDACi treatment (Stimson et al. (2009) Cancer Letters 280:177-183).

However, this signature is likely to vary according to tumour type as well as duration of treatment, and inhibitor concentration. Further, these signatures are response signatures rather than prognostic signature of the drug effect.

Thus, to date, no biomarker has been found that could help predict clearly the tumour response to a HDACi (Stimson et al. (2009) Cancer Letters 280:177- 183).

Accordingly, there is a need for such a method.

MicroRNAs are a class of endogenously expressed small non-protein- coding RNAs that can post-transcriptionally modulate the expression of hundreds of genes by inhibiting the translation or promoting the degradation of targeted RNA, thereby controlling a wide range of biological functions such as cellular proliferation, differentiation, and apoptosis. Recent evidence indicates that microRNAs may function as oncogenes or tumor suppressors, and alteration in microRNA expression may play a critical role in tumorigenesis and clinical outcome (Calin et al. (2006) Nature reviews 6(1 1 ):857-66, Esquela-Kerscher et al. (2006) 6(4):259-69).Thus, several microRNAs were reported to be associated with the clinical outcome of chronic lymphocytic leukaemia, lung and colon adenocarcinoma, and breast, pancreas and thyroid cancers.

Summary of the invention

The present invention arises from the unexpected finding, by the inventors, that in cancer cell lines treated with histone deacetylase inhibitors, the expression of miR-129-5p and miR-133b is specifically increased and that this increase is responsible for the induction of cell cycle arrest which is observed after treatment.

Thus, the present invention relates to a method, in particular an in vitro method, for determining the anti-cancer activity of a compound comprising:

i) measuring the level of expression of a mi-RNA selected from miR-129-5p and/or miR-133b and/or precursors thereof in cancer cells which have been in contact with said compound; ii) comparing the level of expression with a reference value;

iii) determining therefrom the anti-cancer activity of the compound.

The present invention also relates to at least one nucleic acid

(i) comprising or consisting of, or

(ii) encoding a nucleic acid comprising or consisting of,

a sequence selected from the group consisting of:

1 ) SEQ ID NO: 1 and SEQ ID NO: 2 and

2) a sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 by substitution, deletion or insertion of at least one nucleotide, provided that a nucleic acid consisting of the sequence derived from SEQ ID NO: 1 and SEQ

ID NO: 2 is liable to induce cell cycle arrest in cancer cell,

for use as a medicament, in particular for treating cancers.

The present invention also relates to a pharmaceutical composition comprising as active ingredient substance at least one nucleic acid

(i) comprising or consisting of, or

(ii) encoding a nucleic acid comprising or consisting of,

a sequence selected from the group consisting of:

1 ) SEQ ID NO: 1 and SEQ ID NO: 2 and

2) a sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 by substitution, deletion or insertion of at least one nucleotide, provided that a nucleic acid consisting of the sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 is liable to induce cell cycle arrest in cancer cell,

optionally in association with a pharmaceutically acceptable carrier.

The present invention also relates to a method for treating cancer in a patient, comprising administering the patient with a therapeutically effective quantity of at least one nucleic acid

(i) comprising or consisting of, or

(ii) encoding a nucleic acid comprising or consisting of,

a sequence selected from the group consisting of:

1 ) SEQ ID NO: 1 and SEQ ID NO: 2 and

2) a sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 by substitution, deletion or insertion of at least one nucleotide, provided that a nucleic acid consisting of the sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 is liable to induce cell cycle arrest in cancer cell.

Brief description of the drawings

Figure 1 represents modifications in microRNA miR-133b expression in thyroid treated cells (TPC1 , BcPAP, 8505c, CAL62) after TSA incubation (18h, 330 nmol/L) analyzed by RTQ-PCR. Results represent the fold increase of miR-133b in treated cells versus untreated used as reference (relative quantification, RQ). Stars indicate a significant increase versus reference.

Figure 2 represents modifications in microRNA miR-129-5p expression in thyroid treated cells (TPC1 , BcPAP, 8505c, CAL62) after TSA incubation (18h, 330 nmol/L) analyzed by RTQ-PCR. Results represent the fold increase of miR-129-5p in treated cells versus untreated used as reference (relative quantification, RQ). Stars indicate a significant increase versus reference.

Figure 3 represents the increase in expression of miR-129-5p and miR-133b in TPC1 cells treated with either TSA (18h, 330 nmol/L, gray bars) or SAHA (18h. 2.5 μΜ, black bars) HDACi. Results represent the fold increase of miR-129-5p and miR-133b in treated cells versus untreated used as reference (relative quantification, RQ). Stars indicate a significant increase versus reference.

Figure 4 represents the modification in expression of miR-129-5p and miR-133b in non-cancerous adenoma (black bars) and hyperplasic (gray bars) cells treated with TSA (18h, 330 nmol/L, gray bars) HDACi. Results represent the fold increase of miR-129-5p and miR-133b in treated cells versus untreated used as reference (relative quantification, RQ).

Figure 5 represents cell survival measured as the percentage of MTT viability (vertical axis) of TCP1 cells transfected with either antagomiR-control (amiR-CON) or antagomiR-129-5p (amiR-129-5p) for 48h and incubated without or with TSA (330 nmol/L) for the last 24h. Stars indicate a significant difference of MTT viability with or without TSA. n.s. not significant.

Figure 6 represents the percentage subG1 cells among TCP1 cells transfected with either antagomiR-control (amiR-CON) or antagomiR-129-5p (amiR-129-5p) for 48h and incubated without or with TSA (330 nmol/L) for the last 24h. Stars indicate a significant difference of subG1 cells with or without TSA. n.s. not significant. Figure 7 represents GALNT1 expression quantification in cells after transfection of antagomiR and incubation without or with TSA (330nmol/L) for the last 24h.

Figure 8 represents cell survival measured as the percentage of viability (vertical axis) of TPC1 (A) or CAL62(B) cells transfected for 24h with either a control miRNA (miRNA-Neg) or miR-129-5p (lipofectamine alone was used as control) and incubated with HAMLET (0.3mg/ml) or etoposide (0.3mM) or staurosporine (0.3μΜ) for 24h. Cell death was followed using a XTT viability assay.

Detailed description of the invention

As intended herein, the expression "compound" means any molecule or combination of molecules liable to be used in a therapeutic treatment of a patient. A compound which has an "anti-cancer activity" relates to a compound according to the invention for use for treating a patient suffering from cancer. Preferably, the compound according to the invention is a compound able to alter chromatin structure. More preferably, the compound according to the invention is selected from a group consisting of a histone deacetylase (HDAC) inhibitor and a DNA methyltransferase inhibitor.

Examples of histone deacetylase inhibitors according to the invention notably encompass hydroxamic acids, such as Trichostatin A (TSA), cyclic tetrapeptides (such as trapoxin B), depsipeptides, benzamides, electrophilic ketones, aliphatic acid compounds such as phenylbutyrate and valproic acid, SAHA (Vorinostat), CAY10433, HDAC1 , CAY10591 , CAY10398, CAY10603, CBHA, HDAC8, M344, Salermide, Sodium Butyrate, Belinostat/PXD101 , MS275, LAQ824/LBH589, CI994, and MGCD0103. Other histone deacetylase inhibitors according to the invention are also described in Wang & Dymock (2009) Expert Opin. Ther. Pat. 19:1727-1757.

Preferably, the HDAC inhibitor according to the invention is selected from the group consisting of Trichostatin A (TSA), SAHA (Vorinostat), CAY10433, HDAC1 , CAY10591 , CAY10398, CAY10603, CBHA, HDAC8, M344, Salermide and Sodium Butyrate. More preferably, the HDAC inhibitor according to the invention is selected from the group consisting of Trichostatin A and SAHA (Vorinostat).

Methyltransferases catalyze methylation of DNA at position 5 on cytosine.

Aberrations in methylation play a causal role in a variety of diseases, including cancer. Methyltransferase inhibitors inhibit the action of methyltransferases. As an example of methyltransferase inhibitors according to the invention, one can cite 5- Aza-2'-deoxycytidine (5-Azadc), 5-azacytidine (Vidaza), N-acetyl-S-farnesyl-L- cysteine, S-Farnesyl Thioacetic Acid, N-acetyl-S-geranygeranyl-L-Cysteine, Phenylethanolamine N-methyltransferase, Catechol-O-methyl transferase, Histamine dihydrochloride, Tryptamine hydrochloride, Hydralazine hydrochloride, Procainamide hydrochloride, azacytidine, decitabine, entecapone, chaetocin, SKF 91488 dihydrochloride, S-Farnesylthiacetic acid, 5-AzadCyd, RG108, Zebularine, 2'-deoxycytidine, arabinosylcytosine (Ara-C), arabinosyl-5-azacytosine (fazarabine), dihydro-5-azacytidine (DHAC).

The terms "cancer" and "malignancy" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. In particular, the cancer may be thyroid cancer, skin cancer like melanoma, breast cancer, bladder cancer, uterine/cervical cancer, oesophageal cancer, pancreatic cancer, colon carcinoma, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, non-small cell lung cancer and stomach cancer, B or T cell malignancy, leukaemia.. Preferably, the cancer according to the invention is selected from the group consisting of thyroid cancer, skin cancer, for example melanoma, or colon carcinoma. More preferably, the cancer according to the invention is thyroid cancer or colon carcinoma.

According to the invention, the term "patient" or "individual" to be treated is preferably intended for a human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate) affected or likely to be affected with cancer. Preferably, the patient is a human.

The term "treating" or "treatment" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in destroying or depleting cancer cells. Most preferably, such treatment leads to the complete depletion of cancer cells. Preferably, the depletion of the cancer cells is obtained by inducing an arrest of the cellular cycle of said cells.

As intended herein, the expression "cellular cycle" relates to the series of events that take place in a cell leading to its division and duplication (replication). As is well known, the cycle is divided in four successive phases G1 , S, G2 and M. The expression "an arrest of the cellular cycle" means that this cycle is totally stopped in either one of the phases. Such an arrest leads to cellular death.

The "cells from cancer", "cancer cells" or "cancerous cells" according to the invention can originate from a cancer cell line. For example, when the efficiency of a compound for the treatment of thyroid cancer is tested, the cancer test cells may be the papillary thyroid cell lines CAL62, 8505c, BcPAP and TCP1 , notably described by Schweppe et al. (2008) The Journal of clinical endocrinology and metabolism 93(1 1 ):4331 -41 . The methods and techniques for growing these cells depend on the types of cells which are being tested and are well known by the skilled person.

Alternatively, the "cells from cancer" "cancer cells" or "cancerous cells" according to the invention can be cells from a patient affected with cancer, preferably a cancer according to the invention. The patient can optionally be under anti-cancer treatment, preferably under treatment with the compound according to the invention. More preferably, the cancer cells according to the invention are cancer cells from a patient treated with the compound according to the invention.

As intended herein, the expression "cancer cells which have been in contact with said compound" preferably means that the cancer cells have been contacted with the compound which anti-cancer activity is to be determined under conditions and concentrations which allow the compound to have an effect on the cells. These conditions and concentrations can be easily be determined by the person skilled in the art.

The contact between the cancer cells and the compound which anti-cancer activity is to be determined can be an in vitro contact and/or an in vivo contact between the cancer cells and the compound.

An in vivo contact according to the invention notably occurs when the cancer cells are cells from a patient treated with said compound. In this case, the level of expression of a mi-RNA selected from miR-1 29-5p and/or miR-133b and/or precursors thereof can be either directly detected in the cells or be determined upon a further in vitro contact with compound.

By way of example of an in vitro contact, cells from a cancer can be contacted with trichostatin A (TSA) at a concentration of about 100ng/ml for about 10 hours.

The term "mi-RNA" or "microRNA" is used to qualify a class of RNAs generally 10 to 30, in particular 15 to 25 nucleotides long, and play a role in the post-transcriptional regulation of specific genes, by degrading or blocking the translation of the mRNAs resulting from the transcription of these genes. In particular, it is preferred that mi-RNAs according to the invention are mi-RNAs which expression is increased after administration of an anti-cancer medicament to a patient, which increase in expression can further be involved in the anticancer effect of this medicament. Preferably, the mi-RNAs according to the invention are miR-129-5p, in particular Homo sapiens (hsa) miR-129-5p, more particularly represented by SEQ ID NO: 1 , and miR-133b, in particular Homo sapiens (hsa) miR-133b, more particularly represented by SEQ ID NO: 2.

As intended herein, the terms "miR-129-5p" and "miR-133b" notably encompass all the various allelic or polymorphic variants of miR-129-5p, in particular of hsa miR-129-5p, and of miR-133b, in particular of hsa miR-133b, as well as the various orthologous sequences of miR-129-5p and miR-133b, i.e. the species-specific sequences.

miR-129-5p and miR-133b are notably described in Griffiths-Jones (2004)

Nucleic Acids Res 32:D109-D1 1 1 ; Griffiths-Jones et al. (2008) Nucleic Acids Res 36:D154-D158; and the miRBase database available at http://microrna.sanger.ac.uk. A "precursor" of a mi-RNA according to the invention refers to any intermediate in the mi-RNA maturation pathway starting from the transcript obtained from the transcription of a chromosome and ending with the obtaining of a mi-RNA. Thus, a precursor according to the invention can be a mi-RNA primary transcripts (pri-miRNA) or a pre-miRNA, as well as a long non-coding RNA, a snoRNA or a transposon which encodes a mi-RNA before processing via the enzyme Dicer. The most preferred precursor of miR-129-5p according to the invention is miR-129-2, in particular represented by SEQ ID NO: 3. The most preferred precursor of miR-133b according to the invention is miR-133b hairpin, in particular represented by SEQ ID NO: 4. miR-129-2 and miR-133b hairpin are notably described in Griffiths-Jones (2004) Nucleic Acids Res 32:D109-D1 1 1 ; Griffiths-Jones et al. (2008) Nucleic Acids Res 36:D154-D158; and the miRBase database available at http://microrna.sanger.ac.uk.

Particularly preferred variants of miR-129-5p and of miR-133b may lack one or two nucleotides on the 3' end with respect to their respective consensus sequences, in particular represented by SEQ ID NO: 1 and 2. Other particularly preferred variants of miR-129-5p and of miR-133b may comprise one or two additional nucleotides with respect to their respective consensus sequences, in particular represented by SEQ ID NO: 1 and 2, said additional one or two additional nucleotides being those immediately on the 3' end of the sequence of miR-129-5p and miR-133b within the sequence of their respective precursors, miR-129-2 and miR-133b hairpin, which are themselves notably represented by SEQ ID NO: 3 and 4 respectively.

The level of expression of the mi-RNAs in the cells can be measured by any techniques known by the person skilled in the art. Numerous methods are available which allow quantifying a target RNA, for example, methods based on PCR after reverse transcription (RT-PCR) using oligonucleotides which are specific of the target RNA sequences, or alternatively, methods allowing the hybridization of the target RNA, of duplicates or triplicates of the target RNA with probes under stringent conditions. The probes according to the invention are preferably laid down on microarrays. As intended herein, the expression "hybridization under stringent conditions" indicates that the target RNA or the duplicates thereof can specifically bind pairwise, essentially by forming Watson- Crick-type pairs {e.g. G-C pairs or U-A pairs), with probes having sequences complementary thereto. Adequate stringent conditions according to the invention can be easily determined by one of skill in the art. Preferred stringent conditions according to the invention comprise a hybridization step of 10 to 20 hours, preferably 16 hours, at about 40 to 55 °C, preferably 50 °C, under an ionic strength equivalent to that provided by 500 mM to 2 M NaCI, preferably 1 M NaCI. Additional compounds well known to one skilled in the art can also be added such as pH buffers (e.g. Tris or MES), EDTA, Tween, Bovine Serum Albumin, and herring sperm DNA.

The "reference value" according to the invention can be a unique value such as a given level of expression of a mi-RNA or of a precursor thereof, alternatively, it can an average between expression levels of a mi-RNA or of a precursor thereof.

For example, the reference value can be the level of expression of miR- 129-5p and/or miR-133b and/or precursors thereof in cells which are non-cancer cells, which are preferably of the same type as the cancer cells which have been contacted with the compound which anti-cancer activity is determined according to the invention. For example, if the cells from cancer originate from a papillary thyroid cancer, then the non-cancer cells will be non-cancer papillary thyroid cells.

Alternatively, the reference value according to the invention can be the level of expression of miR-129-5p and/or miR-133b and/or precursors thereof in cancer cells which have not been in contact with the compound which anti-cancer activity is determined according to the invention.

As intended herein, the expression "comparing the levels of expression" means that the value of the level of expression of miR-129-5p and/or mi-R133b and/or precursors thereof in the cancer cells after contact with the compound which anti-cancer activity is determined according to the invention is compared to the reference value for, respectively, miR-129-5p and/or mi-R133b and/or precursors thereof.

The expression "determining the anti-cancer activity" as used herein relates to the identification of compounds likely or unlikely to have an effect in the treatment of cancer. As intended herein, the expression "increase in the level of expression of mi-RNA selected from miR-129-5p and/or mi-R133b and/or precursors thereof" indicatess that the level of expression is higher in the cancer cells after treatment with the compound according to the invention, compared with the reference value.

Preferably, an increase of the level of expression indicates that the compound presents an anti-cancer activity. Preferably, as intended herein, the level of expression of miR-129-5p or mi-R133b or precursor thereof is increased in the cancer cells contacted with compound when the level of expression is at least 1 .2 fold, more preferably at least 1 .5 fold, and most preferably at least 2 fold over the reference value.

The method of the invention may further comprise monitoring the level of expression of a mi-RNA selected from miR-129-5p, miR-133b and precursors thereof over a period of time in cancer cells from a patient treated with the compound which anti-cancer activity is determined according to the invention. Preferably, the anti-cancer activity of a compound used for treating cancer in a patient treated with the compound is determined according to the invention at consecutive times during the course of the treatment in order to establish a therapeutic follow-up of the patient. The follow-up is typically performed by determining the level of expression of miR-129-5p, miR-133b and precursors thereof according to the invention, at various time intervals, for instance every 2 weeks, 1 month, 2 months, 3 months, 5 months, etc.

Thus, when a therapeutic follow-up is performed, the reference value according to the invention can also be, in addition to the previous definitions of the term, a previous level of expression of a mi-RNA selected from miR-129-5p and/or miR-133b and/or precursors thereof in cancer cells form the same patient.

Preferably, the therapeutic follow-up according to the invention is started at the same time or upon onset of the treatment of the patient with an anti-cancer compound according to the invention. Advantageously, a decrease in the level of expression of the mi-RNA selected from miR-129-5p and/or miR-133b and/or precursors thereof during the therapeutic follow-up is preferably indicative of a decrease of the anti-cancer activity of the compound thereby indicating a loss of sensitivity of cancer cells the patient to the compound. As intended herein the nucleic acid for use as a medicament, comprised in a pharmaceutical composition, or administered to a patient, can be of any type, it can notably be natural or synthetic, DNA or RNA, single or double stranded. In particular, where the nucleic acid is synthetic, it can comprise non-natural modifications of the bases or bonds, in particular for increasing the resistance to degradation. Where the nucleic acid is RNA, the modifications notably encompass capping its ends or modifying the 2' position of the ribose backbone so as to decrease the reactivity of the hydroxyl moiety, for instance by suppressing the hydroxyl moiety (to yield a 2'-deoxyribose or a 2'-deoxyribose-2'-fluororibose), or substituting the hydroxyl moiety with an alkyl group, such as a methyl group (to yield a 2'-0-methyl-ribose).

In the frame of the above-defined medicament, pharmaceutical composition and method of treatment, where the nucleic acid of the invention comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 2, or of the sequences derived therefrom, the nucleic acid of the invention is intended to directly exert its effect on its cellular targets. In this case, the nucleic acid is preferably a RNA molecule. In contrast, where the nucleic acid of the invention encodes a nucleic acid comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 2, or of the sequences derived therefrom, the nucleic acid of the invention is intended to be expressed within cells where the nucleic acid it encodes, in particular a RNA molecule, will exert its effect on its cellular targets. In this case the nucleic acid of the invention is preferably a DNA molecule, more preferably a double stranded DNA molecule. Besides, as will be clear to one of skill in the art, the nucleic acid according to the invention preferably also comprises genetic elements ensuring expression of the encoded nucleic acid, in particular a promoter sequence of RNA polymerase II or III.

Methods for delivering nucleic acids into cells in vitro or in vivo are well known to one of skill in the art and are notably described in Nguyen et al. (2008) Curr Opin Mol Ther 10:158-67 and Dykxhoorn et al. (2006) Gene Therapy 13:541 - 552, which are incorporated herein by reference.

Preferably, in the frame of the above-defined medicament, pharmaceutical composition and method of treatment, where the nucleic acid of the invention comprises SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence derived therefrom, it is less than 1000 nucleotides long, more preferably less than 100 nucleotides long, and most preferably less than 50 nucleotides long. Also preferably, the sequence derived from SEQ ID NO: 1 or SEQ ID NO: 2 by substitution deletion or insertion of at least one nucleotide presents at least 85%, more preferably at least 90%, and most preferably at least 95% identity with the sequence from which it is derived. As intended herein, the percentage of identity between two sequences is obtained by aligning the two sequences so as to maximize the number of positions of each sequence for which the nucleotides are identical and dividing the number of positions of each sequence for which the nucleotides are identical by the number of nucleotides of the longer of the two sequences.

More preferably, the medicament or the pharmaceutical composition according to the invention comprises, or the method of treatment of the invention comprises administering, a RNA molecule consisting of SEQ ID NO: 1 and/or a RNA molecule consisting of SEQ ID NO: 2.

Most preferably, the medicament or the pharmaceutical composition according to the invention comprises, or the method of treatment of the invention comprises administering, miR-129-5p or a precursor thereof, such as miR-129-2, as defined above, and/or miR-133b or a precursor thereof, such as miR-133b hairpin, as defined above.

Where a medicament, a pharmaceutical composition, or a method of treatment of the invention is contemplated, the nucleic acid can be associated to one or more pharmaceutically acceptable carriers. In particular, it is preferred that the pharmaceutically acceptable carrier be suitable for delivering nucleic acid into cells. Carriers suitable for delivering nucleic acid into cells are well known to one of skill in the art and notably comprise cationic lipids or peptides, nanoparticles and liposomes, optionally linked to moieties, such as antibodies or antibody fragments, having a specificity towards a specific receptor of the target cells, notably cancer cells.

Either local or systemic routes can be used for administering the nucleic acid of the invention to a patient. Examples of administration procedures for nucleic acids are notably described in Nguyen et al. {op. cit.) and Dykxhoorn et al. {op. cit.)

Preferably, a RNA molecule consisting of SEQ ID NO: 1 and/or a RNA molecule consisting of SEQ ID NO: 2 according to the invention are administered to a patient in need thereof in combination or are present together in a same medicament or pharmaceutical composition.

Preferably also, the at least one nucleic acid according to the invention is combined in a medicament or a pharmaceutical composition, or administered in combination, with at least one other anti-cancer compound. In particular, the other anti-cancer compound can be a medicament useful to treat thyroid cancer or skin cancer like melanoma cancer. Examples of such medicaments include cytotoxic agent such as gemcitabine, paclitaxel, cisplatin, etoposide and doxorubicin and mechanism-based agents such as HSP90 inhibitor17-AAG and the proteasome inhibitor bortezomib.

EXAMPLE

1. Materials and Methods 1.1. Reagents and HDACi

Etoposide, Staurosporine and the most widely used HDAC inhibitor Trichostatin A (TSA) were provided by Sigma-Aldrich (Paris, France) and the clinically-relevant HDACi Vorinostat (SAHA) by Cayman Chemical (Ann Arbor, Ml). HAMLET was produced from native purified human milk alpha-lactalbumin on an oleic acid- conditioned ion exchange matrix.

miRNAs (Pre-miR™ miRNA Precursor Molecules, Ambion®) and antagomiRs (Anti-miR™ miRNA Inhibitors, Ambion®) were purchased from Applied Biosystem (Courtaboeuf, France). 1.2. Cell lines and treatment

Thyroid cancer cell lines BcPAP [papillary, BRAF-V⁶⁰⁰E], TPC1 [papillary, RET- PTC1 ], 8505c [anaplasic, BRAF-V⁶⁰⁰E] and CAL62 [anaplasic, KRAS-G¹²R] have been previously characterized (Ito et al. (1993) Cancer research 53:2940-3; Fabien et al. (1994) Cancer 73:2206-12; and Gioanni et al. (1991 ) Bulletin du cancer 78:1053-62).

Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamax, penicillin (100 units/ml)-streptomycin (100 μ9/ιηΙ), sodium pyruvate (1 mM) (Invitrogen, Cergy Pontoise, France), 10% fetal calf serum. The absence of cross-contaminations between the cell-lines was checked by amplification and sequencing of RET/PTC 1 , BRAF-V⁶⁰⁰E, and KRAS-G¹²R after each defreezing (every 2 months) (Schweppe ei al. (2008) Journal of clinical endocrinology and metabolism 93:4331 -41 ).

To test the toxicity sensitivity of HDACi treatments on healthy normal thyroid cells, primary cultures of thyrocytes were derived from adenoma or hyperplasia thyroid tissue immediately after thyroidectomy at the University of Nice hospital. Patient consent was received and the institutional review board approved the project. Cell dissociation procedures, were similar to those used previously. Briefly, thyroid tissue was cut into small pieces (less than 1 mm diameter), washed in PBS and digested for 3x20 min with a solution consisting of 10 U/ml collagenase, 1 mg/ml dispase (Invitrogen) in PBS. After filtration on 300 μιη gaze, cells were seeded at a density of 10⁵ per 25cm² Flask. Cells were fed with F12/Coon's medium supplemented with 5%FCS twice a week.

For all experiments, cells were grown to 70% confluence, and TSA (330nM) or Vorinostat (2.5 μΜ) were added for 16h in fresh complete medium. As controls, cells were left untreated. The effects of HDACi were assessed on cell viability (cell cycle, MTT and morphology) by biochemical assays as well as miRNA and mRNA profiling by microarray analyses.

1.3. miRNA Transfection

Cells were plated at 100 000 cells/well in a 6 wells plate, after 24 h, cells were washed and incubated in 1 ml antibiotic free medium (7 vol: 3 vol DMEM/Optimem) containing 2.5 μΙ of Lipofectamin RNAIMAX, 5 pmol/L of the specific miRNA, or 20 pmol/L of the specific antagomiR. Serum was added after 6 h. When indicated, the following day, HDACi were added in fresh complete medium for 24h. 48h after transfection, cells were lysed for protein or RNA analysis as described in the following parts. Rates of transfection were checked by RT-PCR of the specific transfected miRNA on 7500 thermal cycler (Applied Biosystem).

1.4. Cell cycle analysis

Cell cycle analysis was used in order to analyze both growth arrest (with S phase decrease), and cell death (by increase in subG1 cells) as two endpoints for HDACi activity (Brest et al. (2007) Cancer research 67:1 1327-34). Cells were harvested and fixed in 75% ethanol for 2h. After washes, fixed cells were stained with propidium iodide (2.5 μ9/ιτιΙ, Sigma-Aldrich) and RNase A (250 μ9/ιτιΙ) at 4°C overnight prior to measurement. Cellular fluorescence was measured using the Facscalibur flow cytometry (Becton Dickinson, San Jose, CA). The single-cell populations were determined on the basis of their fluorescence-intensity values FL2-A and FL2-W. S-Phase percent was calculated as a ratio between (S-phase cells)/(G1 +S+G2-phase cells). Cell death quantification was calculated as a ratio between (SubG1 -phase cells)/(subG1 +G1 +S+G2-phase cells). 1.5. Western blot

Cells were lysed in Laemmli buffer (12.5 mM Na₂HP0₄, 15% glycerol, 3% SDS). 50 μg of protein extracts were separated by SDS-PAGE, electrotransferred onto a PVDF membrane, and incubated with anti-acetyl histone H4 (1 /3000, Millipore, Molsheim, France), anti-p21 WAF1 (1 /1000, Santa Cruz Biotechnologies, Heidelberg, Germany), anti-phospho-ser139-H2AX (clone JBW, 1 /3000, Millipore), or anti-PARP (1 /1000, Millipore), in TN-Milk buffer (10 mM Tris HCI, pH 7.4, 0.15 M NaCI, 1 mM EDTA, 0.1 % Tween-20, 5% non fat milk) overnight at 4°C. Appropriate HRP-conjugated secondary antibodies were then applied for 1 h at room temperature. Immunoreactive bands were revealed by enhanced chemiluminescence (ECL, PerkinElmer, Courtaboeuf, France). 1.6. MTT Colorimetric Viability Assay

Cell survival was examined using the MTT colorimetric assay as described (Xiao et al. (2009) Molecular cancer therapeutics 8:350-6). After miRNA transfection and/or HDACi treatment, cells were detached, plated in 96-well plates at 2 10⁴ cells/well in 4-6 replicates, and incubated with 150 μΙ_ MTT (0.5 mg/ml) for 4 h at 37 °C. The accumulated formazan in viable cells was dissolved in 100 μΙ_ DMSO, and then quantified by optical density at 570 nm (Dynex, France). Cell viability was estimated as a percentage of the value of untreated controls (100%). All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average value ± SD of representative experiments.

1.7. Total RNA Extraction

After incubations, cells were lysed in Trizol (Invitrogen). After chloroform, water fraction containing total RNA was collected. After addition of absolute ethanol (1 .5vol/vol), total RNA was purified with the RNAeasy extraction kit (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions. The quality was assessed by measuring both the optical density (Nanodrop, Wilmington, DE), and the electrophoretic mobility using a Bioanalyzer (Agilent Technologies, Santa Clara, CA).

1.8. Global miRNA Expression profiling induced by HDACi

The transcriptional profile induced by HDACi was characterized by oligonucleotide microarray analysis of cells treated with HDACi (1 6 h) vs. control cells, by using a microarray containing 2054 mature miRNAs (409 homo sapiens) found in the miRNA registry, as described by Potier et al. (2009) PloS One 4:e671 8. Fragments of tRNAs, snoRNAs, 5S and 5.8S RNA were also printed on the microchip, providing internal positive controls for specific hybridization. The oligonucleotide sequences are available on http://www.microarray.fr/microRNA/. Each oligonucleotide was spotted four times on each slide (2 distinct pairs of spots), in order to reduce positional bias of the fluorescence readout. This miRNA platform has been registered on the Gene Expression Omnibus public data repository (GEO) under the reference GPL471 5.

Total RNA were labeled with Cy3 or Cy5 fluorescent dye using the Ulysis Alexa fluor nucleic acid labeling kit (Amersham Bioscience, Pittsburgh, PA), then miRNA were isolated using the mirVana miRNA isolation Kit (Applied Biosystem). Three independent experiments were made to identify miRNA up regulated or down regulated in cells. For each oligonucleotide microarray, TI F images containing data from each fluorescence channel were quantified with GenePix Pro 6.1 program (Axon Instruments). Normalization was obtained with the software limma from Bioconductor according to the vsn approach described by Huber et al. (2002) Bioinformatics 18 Suppl 1 :S96-1 04.

1.9. Microarray Analysis of Gene Expression profile induced by miRNA transfection

The transcriptional profile induced by miRNA transfection was characterized by oligonucleotide microarray analysis of cells treated with miR-1 33b or miR-1 29-5p vs. control miRNA transfected cells, by using oligonucleotide microarrays containing 25.484 distinct oligonucleotide probes covering most of the known human transcripts (Le Brigand et al. (2006) Nucleic acids research 34:e87). The list of the probes (length 51 bp) is available online htjp:/ www.irilcroarray.jr. Microarrays were printed with a ChipWriter Pro (Bio-Rad) on commercial hydrogel slides (Schott) and processed according to the manufacturer's instructions.

Total RNA (2 μg) were amplified with the Amino Allyl MessageAmp aRNA kit (Ambion) according to the manufacturer's instructions. Cy3- and Cy5-labeled aRNA were hybridized on array for 17 h at 62 °C (a dye-swap method has been used). Arrays were then washed with expression wash buffer kit (Agilent) and were scanned with a genePix4000B microarray scanner (Axon instrument).

16-bit TIF images were quantified with the corresponding software (GenePix Pro 6.1 program (Axon Instruments) for the GenePix and Quantarray for the ScanArray. Intra and inter slide normalization of three independent experiments were performed using Global Loess and the quantile methods respectively. Means of ratios from all comparisons were calculated and B test analysis was performed using the Limma package available from Bioconductor (Gentleman et al. (2004) Genome Biol 5:R80). Differentially expressed genes were selected using a Benjamini-Hochberg correction of the p-value for multiple tests, with a positive B value. Before analysis, the sylamer algorithm was used in order to characterize the specificity of miR-133b and miR-129-5p-transfection on global mRNA gene expression. As expected, mRNA harboring the seed sequences of miR-133b and miR-129-5p were specifically downregulated. The human targets of the differentially expressed miRNAs were predicted using public Web-based prediction tools, such as PicTar (http/./pictar.bio.nyu.edu) (Krek et al. (2005) Nature genetics 37:495-500), TargetScan (http://cjenes.rnit.edu/tarqetscan/index.htmj) (Lewis et al. (2005) Cell 120:15-20), and miRBase Targets

(ht;p: microrna.sarKjer ac. jk-^';arqet$/v3/) (Griffiths-Jones et al. (2006) Nucleic acids research 34(Database issue):D140-4).

1.10. Validation of miRNA and pangenomic arrays

For validation of mRNA putative target identified by microarray, mRNA from HDACi treated- or miR-133b and miR-129-5p transfected- cells were subjected to reverse transcription-polymerase chain reaction followed by specific Taqman assays (Applied Biosystems). Relative fold changes of expression in induced samples against control were calculated using the comparative Ct (2-ΔΔΟί) method. Real-time quantization was carried out on the 7500 Fast (Applied Biosystem) thermal cycler, and reagents provided by Applied Biosystem under conditions suggested by the manufacturer.

1.11. Statistical analysis

For statistical analysis, GraphPad Instat3 program was used. The results were evaluated for statistical significance by student t-test or ANOVA test. Error bars were marked as the standard deviation (S.D.) of the mean, p values less than 0.05 were regarded as significant. 1.12. Viability assay

Cell survival was examined using the Cell Proliferation Kit II (XTT) from Roche and quantification of DNA fragmented-subG1 population by cell cycle analysis as previously described (19). All experiments were repeated at least three times, and each XTT experimental sample (5000 cells/well) was repeated at least in quadruplicate wells for each experiment. The data are average values ± SD of representative experiments.

2. Results 2.1. HDACi induce cell cycle arrest and cell death

HDACi increase histone acetylation by preventing HDAC activity (Gorisch et al. (2005) J Cell Sci 118:5825-34). The activity of HDACi on a panel of thyroid cancer cell lines carrying different oncogenes RET/PTC 1 [TPC1 ], BRAF V⁶⁰⁰E [BcPAP, 8505c] and KRAS G¹²R [CAL62], was therefore analyzed by western blotting using specific antibodies for acetylated histone H4. The inventors showed that all thyroid cell-lines used were sensitive to TSA (330 nmol/L), as evidenced by the increased histone H4 acetylation. Moreover, P21 WAF1 (CDKN1 A), a gene down-regulated in cancers (Kastan & Bartek (2004) Nature 432:316-23; Massague (2004) Nature 432:298-306), and previously shown to be restored by HDACi treatment (Brest et al. (2007) Cancer research 67:1 1327-34; Blagosklonny et al. (2002) Molecular cancer therapeutics 1 :937-41 ) was upregulated in HDACi-treated thyroid cells, in agreement with previous studies (Mitsiades et al. (2005) Clin Cancer Res 11 :3958- 65; Luong et al. (2006) Clin Cancer Res 12:5570-7). P21 WAF1 acts as a potent cyclin-dependent kinase inhibitor at the G1 checkpoint causing a rapid G1 -S arrest. Consistently, the inventors found a decrease in S phase in cycling cells (5.8% in TSA-treated cells vs 12.8% unteated cells, p<0.05) and an increase of apoptotic cells with fragmented DNA (66% in TSA treated cells vs 2% in control, p<0.001 ). Furthermore, DNA damage and apoptotic cell death were confirmed by phosphorylation of histone H2AX and by cleavage of PARP with a higher sensitivity of papillary thyroid cells. Altogether these findings indicate that TSA was able to induce cell cycle arrest and apoptotic cell death in thyroid cancer cells, regardless of the oncogenic status.

2.2. HDAC inhibitors induce miR-133b and miR-129-5p overexpression

The molecular mechanism responsible for HDACi antitumoral activity remains elusive. miRNAs are candidates that can be considered as putative tumor suppressor. So far, the relationship between miRNA induction and HDACi sensibility of thyroid tumor cells is unknown. To gain insight into their possible involvement, miRNA profile was assessed by microarray analyzes after HDACi treatment. BCPAP and 8505c cell lines were treated with TSA overnight (330 nmol/L), miRNA were isolated and their expression level was analyzed on microarray (Table 1 ). Among the HDACi-induced miRNAs, the inventors chose to focus their attention on the specific increase in miR-133b and miR-129-5p since their increase was confirmed by RT-PCR after TSA treatment in all the thyroid cell lines used. TSA treatment induced an increase in miR-133b expression ranging from 3-fold (8505c, CAL62) to 5.6-fold (TCP1 ) (Figure 1 ) and an increase in miR- 129-5p expression ranging from 7-fold (CAL62) to 18-fold (TCP1 ) (Figure 2) in comparison with untreated cells used as reference. These results were confirmed by using SAHA, another HDACi, on TCP1 cells that showed similar fold of increase as TSA (Figure 3). Interestingly, the highest induction was found in papillary cell lines and correlated to the higher sensitivity in cell death. At that stage it was of interest to ascertain the specificity of HDACi-induced responses (viability, miRNA expression) on normal healthy thyrocytes. As shown in Figure 4, the increase of miR-133b and miR-129-5p under HDACi treatment was restricted to cancer cells since no significant modification of the level of expression was observed in hyperplasia or adenoma primary thyroid culture treated with TSA. Taken together, these findings clearly indicate that HDACi anti-tumoral activity was closely correlated with their ability to upregulate the expression of miRNA miR- 133b and miR-129-5p: both miRNAs were dramatically induced in the HDACi- hypersensitive TCP1 cells while unchanged in HDACi-resistant healthy cells.

2.3. miR-133b and miR-129-5p overexpressions are sufficient for cell growth arrest

To gain insight into the possible involvement of the miR-133b and miR-129-5p in the mediation of the HDACi-antitumoral effect, these miRNAs were transfected into TCP1 cells. After 48 h, transfected cells were analyzed for cell cycle distribution. Introduction of miR-133b and/or miR-129-5p caused a significant reductions of population of cells entering in S-Phase (5.1 ±0.5% for miR-133b, 3.3±0.4% for miR-129-5p, p<0.05), similarly to HDACi treatment (TSA, 330nM, 24h). Cells transfected with negative control miR-CON1 showed the same number of cells in S-Phase (9.7±0.8%) than non-transfected cells. These data underscore that expression of miR-133b and miR-129-5p are sufficient alone for cell growth arrest in an HDACi-treatment similar manner.

2.4. miR-133b and miR-129-5p overexpressions induce decrease expression of cyclin B1 and cyclin F

This conclusion was strengthened by the fact that genes involved in cell cycle were downregulated by overexpression of these miRNAs. To delineate the putative targets of these miRNA, the mRNA profile of miRNA-transfected TPC1 cells was analyzed by a pan-genomic microarray. The inventors focused on genes containing a seed match for miR-133b and miR-129-5p that were downregulated with a logarithm fold change higher than 0.8. As defined by the Gene Ontology Consortium (www.geneontology.org) (Ashburner et al. (2000) Nature genetics 25:25-9), the miRNA transfection associated-meta-signature mostly shed light on genes involved in the cell division, mitosis, and cell cycle (Tables 2, 3, 4) underlying the involvement of these miRNAs in cell cycle arrest.

In order to confirm putative downregulation of cyclin targeted by miR-133b and miR-129-5p, the expression of cyclin B1 (CCNB1 ) and cyclin F (CCNF) was analyzed by qPCR. Consistently, transfection of either miR-133b or miR-129-5p induced a decrease in cyclin B1 and in cyclin F expression (5.6-fold and 7-fold respectively) similarly than TSA-treated cells. In conclusion, the inventors found that miR-129-5p, miR-133b and HDACi shared similar targets like cyclins B1 and F to induce cell cycle arrest.

2.5. miR-133b and miR-129-5p are not necessary for cell growth arrest

Moreover, the role of miR-133b and 129-5p in HDACi-induced cell cycle arrest was then evaluated by transfection of miRNAs or antagomiRs. Interestingly, an additional effect on S-phase decrease was observed when HDACi and miR-133b or miR-129-5p were combined, showing that both miRNA improved HDACi treatment. Unexpectedly, knocking down miR-133b and/or miR-129-5p expression with antagomiRs did not affect HDACi-induced cell growth arrest, showing a potential compensatory regulation of miR-129-5p and miR-133b knockdown.

In conclusion, HDACi-induced overexpression of miR-129-5p and miR-133b participates to cell cycle arrest via a specific decrease in cyclin B1 and F expression in thyroid cancer cells.

2.6. miR-129-5p is sufficient for HDACi induced cell death

HDACi has been shown to induce cell cycle arrest followed by cell death in thyroid cancer cells. The role of both miR-133b and miR-129-5p was investigated in this context.

Interestingly, miR-129-5p-transfected TCP1 cells showed an increased cell death, suggesting that this miRNA is cytotoxic for cancer cells. The inventors then analyzed pan-genomic microarray of miR-129-5p-transfected TPC1 cells on cell death-involved genes expression. Data collected from three independent biological experiments, revealed that a total of 224 transcripts were significantly modulated (102 up and 122 down-regulated, p<0.01 ) following pre-miR-129-5p transfection when compared to the control condition. Analysis of this signature with Ingenuity Pathway™ software indicated a significant enrichment for "Molecular functions" with terms such as "Cellular Compromise", "Cellular Growth and Proliferation", "Cell Cycle" or "Cell Death". Using the bioinformatic tool "MicroToptable", the inventors then looked for potential over representation of miR-129-5p-predicted direct targets among the downregulated transcripts (cut-offs equal to 8.0 for the log₂ (signal), to -1 for the log₂ (ratio), and to 0.01 for the adjusted p-value) and isolated 42 transcripts corresponding to putative direct targets (Table 6).

Interestingly, the inventors found a decreased expression of GALNT1 (2^{"2 0}~-4 fold), a gene previously described to be regulated by miR-129-5p, and BCL6 (B- cell CLL/lymphoma 6, 2^{"2 4}~-5.3 fold), a repressor of the expression of the pro- apoptotic protein PDCD2 (programmed cell death 2) which was increased in the inventor's model (2^{1 7}~3.2 fold) (Table 5). Of particular interest, BCL6 was previously described to be repressed by HDACi both transcriptionally and post- translationally (Saito et al. (2006) Cancer cell 9:435-43; Bereshchenko et al. (2002) Nature genetics 32:606-13).

These results showed that the BCL6/PDCD2 couple is regulated by miR-129-5p which may be involved in HDACi induced cell death.

2.7. miR-129-5p is necessary for HDACi induced cell death

To examine if miR-129-5p is mandatory for HDACi-induced cell death, TCP1 cells were transfected 24h either with antagomiR-CON1 (control) or antagomiR-129-5p and then incubated with TSA (330 nmol/L, 24h). Moreover, antagomiR-133b alone or combined with antagomiR-129-5p were also used to block TSA-induced cell death.

As shown by microscopy and MTT viability (Figure 5), knockdown of miR-129-5p and/or miR-133b expression blocked HDACi-induced cell death in comparison with antagomiR-CON1 transfected cells treated by HDACi. These results were confirmed by the absence of subG1 population in comparison with TSA-treated cells (Figure 6).

Furthermore, HDACi-dependent repression of GALNT1 expression was reverted by antagomiR-129-5p (Figure 7). Similar results were found in BcPAP, Cal62 and 8505c cells transfected with antagomiR-129-5p and treated with TSA.

In conclusion, by blocking expression of miR-129-5p or miR-133b in cells, the inventors showed a prevention of HDACi-induced cell death, thereby showing that miR-129-5p and miR-133b are mandatory for HDACi killing effect on tumor cells. 2.8. miR-129-5p promotes drug-induced cell death

HDACi have previously been shown to induce cell death in response to other cancer drugs like etoposide, cisplatin or HAMLET. To examine if miR-129-5p is a shared target, we transfected TPC1 cells with either miR-129-5p or miR-CTL for 24h before the addition of increasing concentrations of etoposide or HAMLET. Combination with miR-129-5p increased the sensitivity to HAMLET, staurosporine or etoposide (Figure 8). The loss of viability in response to HAMLET (0.3 mg/ml, 24h) was 40% in miR-CTL transfected control cells, but increased to 70% in tumor cells transfected with miR-129-5p. A similar increase in cell death was obtained in cells treated with staurosporine or etoposide. Thus, while miR-129-5p affects tumor cell viability per se, it also improves the sensitivity to different cancer drugs through effects on HDAC function.

Table 1 : Profile of altered miRNA in TSA-treated BCPAP and 8505C cell lines (Fold change > ±0.75, Adjusted p Value <5 10^"3)

Table 2: Putative targeted genes by miR-129-5p found downregulated pangenomic microarray: GO Pathway, gene list

Table 3: Putative targeted genes by miR-133b found downregulated in pangenomic microarray: GO Pathway, gene list

Name ID AveExpr t Fold

BRCA1 672 10.1 -1.0

CCNB1 891 12.9 -1.2

CCNF 899 9.9 -0.9

CD2AP 23607 9.3 -1.6

CDCA8 55143 9.8 -1.1

CENPE 1062 10.3 -1.0

CENPF 1063 11.6 -0.8

CEP55 55165 10.7 -0.9

CETN3 1070 10.0 -1.4

CIT 11113 11.1 -0.9

DSN1 79980 10.2 -1.2

FBX05 26271 9.7 -0.9

HELLS 3070 9.6 -0.9

MAEA 10296 11.9 -1.3

MCM3 4172 10.1 -0.8

MDC1 9656 9.1 -1.1

MKI67 4288 10.1 -1.0

NCAPG 64151 11.3 -1.3

NEK2 4751 9.9 -1.0

NUSAP1 51203 10.8 -1.2

RFC1 5981 10.3 -0.8

RFC5 5985 10.0 -0.9

SMC1A 8243 10.1 -1.1

TOP2A 7153 11.5 -1.4

TPD52L1 7164 12.0 -2.1

TPX2 22974 12.3 -0.9

WTAP 9589 10.7 -0.9 Table 4: Alteration of cell cycle related protein expression by transfection of miR-

129-5 or miR-133b

Table 5: Apoptotic related genes regulated by miR-129-5p

Log Entrez Gene RT-PCR

Symbol RefSeq p-value

Ratio ID Log

BCL6 NM 001706 -2.4 1.03E-02 604 -2.2

TNFSF10 NM 003810 -2.0 1.05E-02 8743

GALNT1 NM 020474 -1.7 8.40E-03 2589 -2.0

RUNX1 NM 001754 -1.6 2.63E-02 861

CTSB NM 147780 -1.6 1.05E-02 1508

NFKBIA NM 020529 -1.6 1.97E-02 4792

NUPR1 NM 012385 -1.5 1.63E-02 26471

FOX01 NM 002015 -1.4 2.30E-02 2308

BMF NM 001003942 -1.4 2.08E-02 90427

EGR1 NM 001964 -1.1 1.92E-02 1958

BAD NM 004322 -1.1 1.60E-02 572

TNFRSF25 NM 003790 -1.1 2.08E-02 8718

TNFRSF10B NM 147187 0.9 2.52E-02 8795

HDAC1 NM 004964 0.9 2.44E-02 3065

TBPL1 NM 004865 1.0 2.53E-02 9519

GADD45A NM 001924 1.0 2.40E-02 1647

BECN1 NM 003766 1.1 1.64E-02 8678

DAPK1 NM 004938 1.2 2.70E-02 1612

PPARG NM 138712 1.3 1.29E-02 5468

EAF2 NM 018456 1.3 2.97E-02 55840

HOXA3 NM 153632 1.5 2.14E-02 3200

ABCB1 NM 000927 1.5 1.05E-02 5243

PDCD2 NM 144781 1.7 2.79E-02 5134 +1.8

CYCS NM 018947 1.7 2.79E-02 54205

CYR61 NM 001554 2.6 2.08E-02 3491 Table 6: List of the mir-129-5p-predicted direct targets down-regulated following miR-129-5p overexpression in TCP1 cells

Symbol Genbank ID Av. Expr. LogFC

CDKN1C 1028 8.8 -1.3

ACTN1 87 12.9 -1.2

GALNT1 2589 9.7 -1.6

TRIP13 9319 9.9 -1.7

DNAJA3 9093 9.6 -1.3

RUNX1 861 10.1 -1.5

ARPC5 10092 12.8 -1.8

ZNF706 51123 11.8 -1.4

CKAP4 10970 13.1 -1.4

DPY19L1 23333 10.4 -1.8

PTMA 5757 11.6 -2.5

AZGP1 563 9.7 -2.2

ITGAE 3682 11.3 -1.1

C11orf54 28970 9.8 -1.3

NPEPPS 9520 11.8 -2.6

CA12 771 10.8 -2.2

VPS26A 9559 12 -2.3

AK2 204 10.4 -1.5

ZMAT2 153527 11.2 -2.1

CIT 11113 10.8 -1.3

PDXDC1 23042 10.8 -1.2

CCNF 899 9.7 -1.1

DNAH11 8701 10.7 -1.2

CDK6 1021 9.8 -2.2

CACYBP 27101 10.4 -1.7

MATR3 9782 12.6 -1.3

RASSF5 83593 9.5 -1.3

LYRM1 57149 10.4 -1.5

VAMP2 6844 10.3 -1.2

C20orf108 116151 8.4 -1.3

ALDH3A2 224 9.6 -1.2

F AM 129 A 116496 13.2 -2

BPTF 2186 10.3 -1.1

THUMPD2 80745 9.9 -1.1

TNFSF10 8743 9 -1.5

EMP1 2012 12.2 -3.8

LBR 3930 11.9 -1.8

KLHL24 54800 9.3 -1.7

CTBP2 1488 9.6 -1.9

GINS3 64785 10.1 -1.3

DTL 51514 10.2 -1.7

SH3KBP1 30011 11.1 -1

Claims

1. An in vitro method for determining the anti-cancer activity of a compound able to alter chromatin structure comprising:

i) measuring the level of expression of a mi-RNA selected from miR-129-5p and/or miR-133b and/or precursors thereof in cancer cells which have been in contact with said compound able to alter chromatin structure;

ii) comparing the level of expression with a reference value;

iii) determining therefrom the anti-cancer activity of the compound able to alter chromatin structure.

2. The method according to claim 1 , wherein the cancer cells are from a patient treated with the compound able to alter chromatin structure.

3. The method according to claim 1 or 2, wherein the cancer cells are contacted with the compound able to alter chromatin structure in vitro.

4. The method according to any of claims 1 to 3, wherein an increase in the level of expression of a mi-RNA selected from miR-129-5p and/or miR-133b and/or precursors thereof compared to the reference value is indicative of the efficiency of the compound able to alter chromatin structure on said cancer.

5. The method according to any of claims 1 to 4, wherein the reference value is the level of expression of miR-129-5p and/or miR-133b and/or precursors thereof in cancer cells which have not been in contact with the compound able to alter chromatin structure.

6. The method according to any of claims 1 to 4, wherein the reference value is the level of expression of miR-129-5p and/or miR-133b and/or precursors thereof in a non-cancer cell.

7. The method according to any of claims 1 to 6, wherein the cancer cells are from a patient treated with the compound able to alter chromatin structure and wherein the anti-cancer activity of the compound able to alter chromatin structure is determined at consecutive times during the course of the treatment in order to establish a therapeutic follow-up of the patient.

8. The method according to claim 7, wherein a decrease of the anti-cancer activity of the compound able to alter chromatin structure during the follow-up is indicative of lost of the sensitivity of the patient cancer cells to the compound able to alter chromatin structure.

9. The method according to claim 9, wherein the compound able to alter chromatin structure is selected from the group consisting of a histone deacetylase inhibitor and a DNA methyltransferase inhibitor.

10. The method according to any of claims 1 to 9, wherein the cancer is selected from the group consisting of the thyroid cancer, skin cancer and colon carcinoma.

11. At least one nucleic acid

(i) comprising or consisting of, or

(ii) encoding a nucleic acid comprising or consisting of,

a sequence selected from the group consisting of:

1 ) SEQ ID NO: 1 and SEQ ID NO: 2; and

2) a sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 by substitution, deletion or insertion of at least one nucleotide, provided that a nucleic acid consisting of the sequence derived from SEQ ID NO: 1 and SEQ ID NO: 2 is liable to induce cell cycle arrest in cells from cancer,

for use as a medicament.

12. The at least one nucleic acid according to claim 12, wherein the medicament comprises a RNA molecule consisting of SEQ ID NO: 1 and/or a RNA molecule consisting of SEQ ID NO: 2.

13. The at least one nucleic acid according to any one of claims 12 to 13, wherein the medicament is used for treating a cancer.

14. The at least one nucleic acid according to claim 14, wherein the cancer is selected in the group consisting of the thyroid or cancer, skin cancer and colon carcinoma.