WO2017207623A1

WO2017207623A1 - Mirna as biomarkers and regulators of cancer stem cells

Info

Publication number: WO2017207623A1
Application number: PCT/EP2017/063143
Authority: WO
Inventors: Stéphanie Renaud; Nicolas Mermod
Original assignee: Université de Lausanne
Priority date: 2016-05-31
Filing date: 2017-05-31
Publication date: 2017-12-07

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of breast cancer, and for the diagnosis of breast cancer, its metastasis, and breast cancer drug resistance.

Description

mi^'RNA as biomarkers and regulators of Cancer Stem Cells

FIELD OF THE INVENTION The present invention relates to methods and pharmaceutical compositions for the treatment of breast cancer, and for the diagnosis of breast cancer, its metastasis, and breast cancer drug resistance.

BACKGROUND OF THE INVENTION

Breast cancer (BC) is the most common cancer in women worldwide, with over 1 .7 million of patients diagnosed yearly, and it remains the first cause of cancer-related death for women in most western countries. Despite the fact that significant therapeutic advances have been achieved, metastatic BC usually remains incurable. In addition, more reliable predictors of the response to specific therapeutic treatments are needed. In particular, neoadjuvant chemotherapy, initially reserved to treat advanced and inflammatory BC before surgical intervention, is not always beneficial (1 -3). To date, it is commonly admitted that additional biomarkers are needed to better predict the failure of neoadjuvant systemic therapies and BC recurrence.

Solid tumor chemoresistance has been proposed to involve a minority of cells within the tumor, called Cancer Stem Cells (CSCs), that are responsible for tumor regrowth after chemotherapy (4). CSCs are thought to be capable of both self-renewal and of differentiation to other cancer cell types. Moreover, there is increasing evidence that CSCs are resistant to chemotherapy and radiation therapy, and thus, that they may mediate the resistance to treatment, in addition to metastasis to distant organs (5-7). Identification of these cells relies on biomarkers as well as on in vitro and in vivo tumor expansion assays. It has been shown that a subpopulation of BC cells, characterized by the expression of biomarkers such as ESA and CD44 and by the lack of CD24, were able to generate tumors in NOD/SCID mice xenografts model from just 200 cells, whereas a 100-fold excess of BC cells lacking these markers remained non-tumorigenic (8, 9). A mammosphere procedure was developed to enrich CSCs from BC cell populations in vitro (10), so as to allow their maintenance and propagation in culture (1 1 - 13). More recently, the expression of aldehyde dehydrogenase (ALDH) has been correlated with the CSC cellular phenotype (14, 15). While these studies indicated that breast cancer stem cells (BCSCs) might be associated with the resistance to

chemotherapy and radiation therapy, further efforts will be required to identify essential regulators of the BCSC cellular phenotype and how they may mediate cancer cell resistance.

During the last decade, microRNAs (miRNA) have emerged as key players in carcinogenesis. The miRNA are small non coding RNA molecules that suppress gene expression, for instance by interacting with the 3'-untranslated region (3'-UTR) of messenger RNA, thereby regulating a variety of biological processes such as cell fate decision and sternness (16, 17). In BC, miRNA dysregulation has been shown to impact cancer occurrence and resistance to treatment (18). For instance, miR-205 was found to be highly expressed in mouse mammary stem or progenitor cells, leading to their expansion and proliferation (19, 20), which was associated with a down regulation of the Let-7 gene (21 ). Consistently, overexpression of Let-7 inhibited cell proliferation, mammospheres formation, BCSC self- renewal and proliferation, as well as tumor formation and metastasis in NOD/SCID mice (22). More recently, miR-200c was proposed to be a tumor suppressor miRNA by down-regulating Bim-1 , thereby reducing clonogenic and tumor-initiation activities of BCSCs (23).

Therefore, the identification of specific miRNA as potential markers and

regulators of CSCs may constitute a promising avenue towards more successful early cancer diagnosis and treatment. SUMMARY OF THE INVENTION

This object has been achieved by providing a method for diagnosing a breast cancer in a subject and for predicting the resistance to chemotherapy in said subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

(b) determining the expression level of one or more miRNA associated with cancer stem cells,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having or developing resistance to chemotherapy. A further object of the present invention is to provide a method for the diagnosis of metastatic cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having metastatic cancer. A further object of the present invention is to provide a method for predicting the metastatic ability of a cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having or developing metastatic cancer.

A further object of the present invention is to provide a method for predicting the metastatic ability of a cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having or developing metastatic cancer. A further object of the present invention is to provide a method of predicting the likelihood of metastasis-free survival of a breast cancer patient, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of metastasis- free survival of a breast cancer patient. A further object of the present invention is to provide an assay for use in a method of the invention, comprising means and/or reagents for determining the expression level of one or more miRNA associated with cancer stem cells a biological sample from said subject. A further object of the present invention is to provide an miRNA inhibitor for the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

A further object of the present invention is to provide a method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising:

(a) providing an inhibitor of one or more miRNA associated with cancer stem cells ; and

(b) administering said miRNA inhibitor to a subject suffering from breast cancer.

A further object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of one or more miRNA associated with cancer stem cells and a pharmaceutically acceptable carrier or diluent.

A further object of the present invention is to provide a composition comprising an inhibitor of one or more miRNA associated with cancer stem cells.

A further object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of one or more miRNA associated with cancer stem cells for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

A further object of the present invention is to provide a kit for performing a method according to the invention, said kit comprising

a) means and/or reagents for determining the expression level of one or more miRNA associated with cancer stem cells a biological sample from said subject, and

b) instructions for use.

A further object of the present invention is to provide a method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising, comprising i) determining the expression level of one or more miRNA associated with cancer stem cells in a biological sample obtained from a subject,

ii) and treating the subject based upon whether an alteration in the expression level of one or more miRNA associated with cancer stem cells in said biological sample, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of breast cancer, breast cancer metastasis, or breast cancer drug resistance in said subject.

DESCRIPTION OF THE FIGURES

Figure 1. Heat maps of differentially expressed miRNAs in chemoresistant mammospheres. (A) Fold change profiling of miRNA in MCF7 cancer cell-derived mammospheres selected to be resistant to 5-FU or Paclitaxel, as compared to unselected MCF7 mammospheres. (B) Fold change profiling of miRNA MCF7 cells mammospheres resistant to 5-FU or Paclitaxel, as compared to non-tumorigenic MCF10A mammospheres.

Figure 2. miRNA-363-3p expression in breast cancer cell lines. (A) Relative miRNA-363- 3p levels of MCF7 cells from adherent cultures, mammospheres and mammospheres resistant to 5-FU or Paclitaxel (n=3; i-test ^**p< 0.01 , ^***p< 0.001 ). (B) Relative miRNA-363-3p levels of MCF7 and MDA-MB-231 total cell populations (unsorted), or sorted by cytofluorometry to be positive or negative for ALDH activity (n=3; i-test, ^** p< 0.01 and ^*** p< 0.001 relative to unsorted populations).

Figure 3. Effect of miRNA-363-3p inhibition in MCF7 cell line. (A) Relative miRNA-363- 3p levels in MCF7 cells stably transfected with a tetracycline promoter- driven expression vector for a negative control miRNA (miRNA-control), for miRNA- 363-3p, or for An miRNA - 363-3p inhibitor (anti-miRNA-363-3p) and for GFP. Cells were cultivated as adherent cultures in the presence of the doxycycline inducer (n=3; i-test, ^*p< 0.05). (B) The effect of miRNA-363-3p inhibition on mammospheres size and number was determined by fluorescence microscopy. The average numbers and standard deviation of spheres were determined from miRNA-363-3p and anti-miRNA-363-3 expressing MCF7 cells (n=5; i-test^***p< 0.001 ). (C) The miRNA-363-3p expression levels and numbers of colonies formed in soft agar was obtained from MCF7 cells expressing the anti-miRNA-363-3p (n=3 or 9; i-test, ^*p< 0.05 and ^***p< 0.001 ).

Figure 4. In vivo effect of miR-363-3p on tumor formation. MCF7 cells were stably transfected with a tetracycline promoter-driven expression vector for a negative control miRNA (miR-control) or for An miRNA -363-3p inhibitor (anti-miRNA-363-3p) and GFP, and transduced with a luciferase expression vector, prior to culture as mamnnospheres in the presence of doxycycline. 50,000 cells were injected

intraductally in mice mammary glands, as indicated, and mice were provided with doxycycline in the drinking water. (A) Tumor growth in vivo was determined from the luciferase signal (nmiRcontrol=4, nmiR-363-3pi=7; i-test, ^*p< 0.05, ^**p< 0.01 ). The embedded panel displays the fold change of miRNA-363-3p levels in MCF7 cells stably transfected with miRNA-control or anti-miRNA-363-3p prior to injection (n=3; t- test, ^*p< 0.05). (B) Pictures of whole glands displaying the fluorescence of injected MCF7 cells from the GFP cDNA co-transcribed with the indicated miRNAs. (C) RT- qPCR quantification of human GAPDH mRNA, EmGFP mRNA and miR-363-3p in glands of mice injected with miR-control or anti-miR-363-3p expressing MCF7 cells (n=5 or 4 for mice injected with miR-control or anti-miR363-3p, repectively (Mann- Whitney test, ^*p< 0.05). (D) RT-qPCR assay of human GAPDH mRNA in lung of injected mice, representing the invasion from human BC cells in the peripheral organ (n=3 or 6 for mice injected with miR-control or anti-miR363-3p, repectively. Mann- Whitney test, ^* p< 0.05).

Figure 5. Expression of miRNA-363-3p in the human tumor biopsies and blood sera of patients enrolled in a neoadjuvant chemotherapy protocol. (A) Box plot summary of the distribution of miRNA-363-3p expression levels in breast tumor tissues of a group of 38 patients before or after chemotherapy, as indicated, and for 9 next-to-tumor control tissues (NxT tissue) from untreated patients (i-test, ^** p< 0.01 ). (B) Box plot summary of the distribution of miRNA-363-3p levels in sera of the same group of BC patients before or after chemotherapy. Grey and white boxes represented the group of 15 patients with low levels of miR-363-3p, and the group of 22 patients with high levels of miR363-3p, respectively. Within each group, levels of miR363-3p before and after chemotherapy were significantly different (Wilcoxon test, ^*** p< 0.001 ). Levels of miR-363-3p before chemotherapy and after chemotherapy, respectively, were also significantly different between the two populations (Mann- Whitney test, ^* p< 0.05 and ^*** p< 0.001 ). Figure 6. Alteration of miRNA levels upon the selection of chemoresistant cells from CSC-enriched populations. MA plot resulting from miRNA microarray data that were quantile normalized based on summarized expression levels of probe sets. The M value represents the log2 Fold Change for a given miRNA probe set, when

comparing treated to untreated cells. The A value of a probe set is the mean of all expression levels, expressed as the log2 of the raw values of all assay conditions. (A) MA plot comparing MCF7 mammospheres versus MCF7 mammospheres resistant to 5-FU. (B) MA plot comparing MCF7 mammospheres versus MCF7 mammospheres resistant to Paclitaxel. miRNA expressed at lower levels in the resistant population are shown in green, whereas miRNA more highly expressed are shown in red. The circled diamonds depict miR-363-3p.

Figure 7. miR-363-3p expression is a marker of breast cancer cells. miR-363-3p level were assessed by RT-qPCR in different breast cancer cell lines and in normal-like MCF10A breast cells grown in adherent conditions.

Figure 8. miR signature in a patient with breast cancer (before chemotherapy), who responded well to the chemotherapy treatment (after chemotherapy), but who did not agree to surgery and had a relapse of the cancer (relapse). As can be seen, elevated levels of some miRNA are associated with the diseased state and have reduced levels upon successfull treatment (miR363-3p, miR142-3p, miR21 -3p), whereas other miRNAs have an opposite behavior, with low levels associated to the diseased state (miR149-5p, miR494-3p) and an increase upon treatment. Collectively, these miRNAs can be, e.g. used to constitute a signature associated with the occurrence of the disease and its response to chemotherapy. DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise/comprising" is generally used in the sense of

include/including, that is to say permitting the presence of one or more features or components. The terms "comprise" and "comprising" also encompass the more restricted ones "consist" and "consisting", respectively. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein the terms " subject", or " patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject suffering from breast cancer. However, in other aspects, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Reference throughout this specification to "one aspect" "an aspect" "another aspect," "a particular aspect," combinations thereof means that a particular feature, structure or characteristic described in connection with the invention aspect is included in at least one aspect of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

The term "treatment" or "treating" means any administration of a composition, pharmaceutical composition, miRNA inhibitor, modified single cell or population of modified cells, etc... of the disclosure to a subject for the purpose of:

(i) preventing the disease, that is, causing the clinical symptoms of the disease not to develop;

(ii) inhibiting the disease, that is, arresting the development of clinical symptoms; and/or (iii) relieving the disease, that is, causing the regression of clinical symptoms.

In the context of the present invention, the disease is breast cancer, breast cancer metastasis, or breast cancer drug resistance.

According to scientific literature, there are four main intrinsic or molecular subtypes of breast cancer that are based on the genes a cancer expresses: i) Luminal A breast cancer is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive), HER2 negative, and has low levels of the protein Ki-67, which helps control how fast cancer cells grow. Luminal A cancers are low-grade, tend to grow slowly and have the best prognosis; ii) Luminal B breast cancer is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive), and either HER2 positive or HER2 negative with high levels of Ki-67. Luminal B cancers generally grow slightly faster than luminal A cancers and their prognosis is slightly worse; iii) Triple-negative/basal-like breast cancer is hormone-receptor negative

(estrogen-receptor (ER) and progesterone-receptor (PR) negative) and HER2 negative; Triple-negative/basal-like breast cancers have a worse prognosis; iv) HER2-enriched breast cancer is hormone-receptor negative (estrogen- receptor and progesterone-receptor negative) and HER2 positive. HER2-enriched cancers tend to grow faster than luminal cancers and can have a worse prognosis.

Surprisingly, the inventors of the present invention have shown that an alteration in the level of one or more miRNA described herein is associated with breast cancer stem cells, thus suggesting that this miRNA signature is linked to breast cancer and in particular to breast cancer drug resistance. Furthermore, experimental alteration of the expression of one or more miRNA described herein in the context of breast cancer impaired breast cancer stem cells establishment, growth and invasion in vivo thus strongly suggesting that this miRNA signature is linked to breast cancer cell metastasis.

Cancer recurrence and resistance to systemic therapies has been attributed to a small set of cells within the tumors called cancer stem cells, because of their stemness properties. Therefore, significant efforts have been devoted to identify new biomarkers that may improve their detection, to identify patients with a higher risk of recurrence, as well as pharmacological effectors that could be used to specifically target these cells within tumors.

Several miRNA have been previously associated with cancer stem cells (CSCs) or chemoresistance (26-30). However, no such marker had been linked to

chemoresistance of CSCs in breast cancer yet, despite the need for improved breast cancer diagnostic markers.

"miRNAs" as used herein, refer to short non-coding oligonucleotides (RNAs) that are among the most abundant class of small RNAs in animals. Other members of the class of small RNAs include small-interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). While miRNAs and piRNAs are endogenously encoded by the cell, siRNAs originate from an extracellular source, i.e. viral RNAs. MiRNAs, siRNAs, and piRNAs are all regulators of post-transcriptional gene expression, and although their mature end- products are structurally almost identical, they are functionally quite different. While siRNAs regulate gene expression by inducing degradation of the target mRNA, miRNAs can also silence gene expression through translational repression. MiRNAs are wide spread in living species. They are expressed in plants, invertebrates and in vertebrates.

The terms "nucleic acid", "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any kind of deoxyribonucleotide (e.g. DNA, cDNA, ...) or ribonucleotide (e.g. RNA, miRNA, ...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g. DNA RNA) polymer, in linear or circular conformation, and in either single - or double - stranded form. These terms are not to be construed as limiting with respect to the length of a polymer and can encompass known analogues of natural nucleotides, as well as nucleotides that are chemically modified in the base, sugar and/or phosphate moieties.

Chemical modifications of said oligonucleotides or nucleotides can comprise

modifications to the nucleobases, the backbone residues, and/or the internucleoside linkers of said oligonucleotides.

Modifications to one or more nucleobases of said oligonucleotides may comprise one or more alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N-6-methyladenine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1 -methyladenine, 1 -methylpseudouracil, 1 - methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyl uracil, 5-methoxy amino methyl-2-thiouracil, -D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2 methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5- oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2,6,diaminopurine, methylpsuedouracil, 1 -methylguanine and 1 -methylcytosine.

Modifications to one or more backbone residues of said oligonucleotides may comprise one or more of the following: 2' sugar modifications such as 2'-O-methyl (2'-OMe), 2'-O- methoxyethyl (2'-MOE), 2'-O-methoxyethoxy, 2'-Fluoro (2'-F), 2'-Allyl, 2'-O-[2- (methylamino)-2-oxoethyl], 2'-O-(N-methylcarbamate); 4' sugar modifications including 4'-thio, 4'-CH2-O-2'-bridge, 4-(CH2)2-O-2'-bridge; Locked Nucleic Acid (LNA); Peptide Nucleic Acid (PNA); Intercalating nucleic acid (INA); Twisted intercalating nucleic acid (TINA); Hexitol nucleic acids (HNA); arabinonucleic acid (ANA); cyclohexane nucleic acids (CNA); cyclohexenylnucleic acid (CeNA); threosyl nucleic acid (TNA); Morpholino oligonucleotides; Gap-mers; Mix-mers; Incorporation Arginine-rich peptides; addition of 5'-phosphate to synthetic RNAs; RNA Aptamers; or any combinations thereof.

Modifications to one or more internucleoside linkers of said oligonucleotides may comprise one or more of the following: Phosphorothioate, phosphoramidate,

phosphorodiamidate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate and phosphoranilidate, or any combinations thereof.

In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T. For example, an miRNA, or miRNA inhibitor according to the invention, can be modified to enhance expression and reduce possible toxicity by including one or more modified nucleoside e.g. using pseudo-U or 5-Methyl- C.

By combining miRNA microarray profiling with the selection of chemoresistant cell populations from breast cancer cell mammospheres, the inventors have surprisingly shown that an alteration in the level of one or more miRNA is associated with cancer stem cells, thus suggesting that this miRNA signature may be linked to chemoresistant CSCs.

This miRNA signature consisted of miR-363-3p, miR-21 -3p, miR142-3p and miR-149- 5p, which were overexpressed in chemoresistant BCSC-enriched populations, whereas miR-630 and miR494 were downregulated. Several of these miRNA had already been linked to pathways involved in cancer diseases. For instance, miR-21 is expressed in most human tumors, and its overexpression was previously correlated with increased cell migration, chemoresistance and poor survival of the patients (31 -35). MiR-149-5p was found to be downregulated in adenocarcinoma of the oesophagus (36). In glioblastoma, inhibition of miR494-3p was found to promote apoptosis (37), and miR142-3p is dowregulated in tumor- infiltrating macrophages (38). In lung cancer, miR- 630 was found to have a bimodal role in the regulation of apoptosis in response to DNA damage in cancer cells (39). Here we found that the most downregulated miRNA of the signature was miR-630, suggesting an implication in the chemoresistance of breast cancer stem cells. Consistently, it was recently shown to be implicated in the resistance to HER-targeting drugs, since its expression could restore the efficacy of those drugs in HER2 over-expressing breast cancer (40). Overall, these observations indicate that the double selection process used in this study allowed the identification of cancer-relevant miRNAs, and that it could significantly enrich tumor cells implicated in cancer

progression and resistance to therapeutic treatments.

As used herein, "one or more miRNA " means "at least one miRNA", e.g. a combination of two, three, four, five or six miRNAs. This combination can comprise, for example, i) one or more miRNAs which were overexpressed in chemoresistant BCSC-enriched populations, or ii) one or more miRNAs which were downregulated in chemoresistant BCSC-enriched populations, or iii) one or more miRNAs which were downregulated in chemoresistant BCSC-enriched populations and one or more miRNAs which were overexpressed in chemoresistant BCSC-enriched populations.

Preferably, the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR494. Most preferably, the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR494, miR142-3p, and miR-149-5p. For example, the one or more miRNA associated with cancer stem cells is selected from the signature (i.e. the combination) comprising i) miR-363-3p, miR494, and miR-149-5p, ii) miR-363-3p and miR494, and iii) miR-363-3p and miR-149-5p.

Whereas miR-363-3p belongs to the oncomir miR-106a-363 cluster implicated in tumorigenesis, its implication in BC had remained unknown yet. This miRNA was identified as the most highly up-regulated miR within cell populations enriched for BCSCs by independent approaches, either upon culture as mammospheres or when selected for expression of the ALDH stem cell marker. miRNA-363-3p was found to be overexpressed in all BC cell lines tested in vitro (example 1), as well as in vivo (example 1), in human tumors transplanted into murine mammary gland, in human BC biopsies, and in their metastasis both in mice and humans. In contrast, it was lowly expressed in non-tumorigenic MCF10A cells or in the tissues surrounding breast tumors in patients, indicating that it is a specific marker of BC tumor cells and a specific CSC marker.

A role for miR-363-3p ability to drive tumor growth in vivo was established in BC- derived cell lines, where its down-regulation decreased human tumor growth within the murine milkducts, as well as metastasis to the lungs. Interestingly, miR-363-3p was also proposed to have an anti-proliferative action on several cancers, including hepatocarcinomas and T-cell lymphomas (42, 43), which may indicate distinct roles for this miRNA in various types of cancers.

The finding that miR363-3p overexpression can mediate breast cancer stem cell maintenance, tumor growth and metastasis correlates well with the finding of elevated levels of this miR in the exosomes obtained from the sera of breast cancer patients (as shown in example 1 ). Indeed, metastasis was observed in a subgroup of BC patients that displayed high initial levels of miR-363-3p in their serum exosomes, and whose miR levels remained high or further increased upon chemotherapy. Thus, high levels of miR-363-3p are indicative of lower chance of cancer eradication by the chemotherapy, and of a poor outcome, in the contact of breast cancer and possibly other cancers.

Therefore a high expression of miR-363-3p upon initial diagnosis, and a further rise during chemotherapy, can thus provide information in terms of tumor aggressiveness, residual disease and increased risk of recurrence after standard treatments. For instance, patients with bad outcomes usually have already high levels of miR-363-3p in exosomes before chemotherapy.

One current concern with neoadjuvant breast cancer therapy is that it may benefit responding patients, whereas it might be useless for non-responding ones. Currently, there is no simple and reliable assay of the response to chemotherapy until surgery, and even the pCR determination currently used does not provide unambiguous conclusions.

The present invention concerns a method for diagnosing a breast cancer in a subject and for predicting the resistance to chemotherapy in said subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having or developing resistance to chemotherapy.

Usually, miR-363-3p, miR-21 -3p, miR142-3p and miR-149-5p, are overexpressed in chemoresistant BCSC, whereas miR-630 and miR494 are downregulated. Preferably, an upregulated expression of miR-363-3p is indicative of the subject having or developing resistance to chemotherapy.

Generally, the chemotherapy is adjuvant or neoadjuvant chemotherapy, and the chemotherapy drug will be selected from the group comprising doxorubicin, carboplatin, cyclophosphamide, epirubicin, fluorouracil (also called 5-fluorouracil or 5-FU), methotrexate, paclitaxel, docetaxel, or a combination of one or more of these drugs.

In many cases also, chemotherapy drugs are given in combination and are known as chemotherapy regimens such as, e.g. FEC, which is a combination of 5-FU, epirubicin and cyclophosphamide or TAC, which is a combination of docetaxel, doxorubicin and cyclophosphamide.

As used herein, the biological sample is selected from the group comprising whole blood, serum, serum exosomes, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, cancer cells. More preferably, the biological sample is whole blood sample or cancer cell sample.

One or more cancer cell sample may be obtained from a subject by techniques known in the art, such as biopsy. The type of biopsy utilized is dependent upon the exact anatomical location from which the sample is to be obtained. Examples include fine needle aspiration (FSA), core-needle biopsy, excisional biopsy, incisional biopsy, and punch biopsy.

The invention further contemplates a method for the diagnosis of metastatic cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having metastatic cancer.

Also contemplated is a method for predicting the metastatic ability of a cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

The present invention also provides a method for predicting the metastatic ability of a cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

Also provided is a method for determining the progression or regression of cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

(b) periodically determining the expression level of one or more miRNA associated with cancer stem cells,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological sample, relative to the level of corresponding to said one or more miRNA determined previously, is indicative of the progression or regression of said breast cancer.

The invention also contemplates a method of predicting the likelihood of metastasis-free survival of a breast cancer patient, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of metastasis- free survival of a breast cancer patient.

In accordance with the present disclosure, an alteration in the level of one or more miRNA that corresponds to an upregulated or a downregulated expression of said one or more miRNA, relative to the level of a corresponding miRNA in a control sample, is indicative of the presence of breast cancer, breast cancer metastasis, or breast cancer drug resistance in the subject.

In one aspect, the level of the one or more miRNA in the biological sample is greater than the level of the corresponding miRNA in the control sample (i.e., expression of the miRNA is "up-regulated"). As used herein, expression of an miRNA is "up- regulated" when the amount of miRNA in a biological sample from a subject is greater than the amount of the same miRNA in a control biological sample.

The increase, or upregulation, of the expression of one or more miRNA in a biological sample according to the methods of the present invention refers, usually, to an increase of the expression of said one or more miRNA equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, as described above. In another aspect, the level of the one or more miRNA in the biological sample is less than the level of the corresponding miRNA in the control sample (i.e., expression of the miRNA is "down-regulated"). As used herein, expression of an miRNA is "down- regulated" when the amount of miRNA in a biological sample from a subject is less than the amount produced from the same miRNA in a control biological sample.

The decrease, or dowregulation, of the expression of one or more miRNA in a biological sample according to the methods of the present invention refers, usually, to a diminution of the expression of said one or more miRNA equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, as described above.

The relative miRNA expression in the control and normal biological samples can be determined with respect to one or more RNA expression standards. The standards can comprise, for example, a zero miRNA expression level, the miRNA expression level in a standard cell line, or the average level of miRNA expression previously obtained for a population of healthy human controls (i.e. cancer-free subject).

Preferably, the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR-494. Most preferably, the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-3p, and miR-149-5p. Even more preferably, the one or more miRNA associated with cancer stem cells is miR-363-3p and one or more miRNA selected from the group comprising miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR-494.

Alternatively, the methods of the present invention can be associated with the detection of one or more prognostic markers or features, including, a marker associated with an adverse (i.e., negative) prognosis, or a marker associated with a good (i.e., positive) prognosis. In certain aspects, the breast cancer that is diagnosed using the methods described herein is associated with one or more adverse prognostic features selected from the group consisting of estrogen receptor expression, progesterone receptor expression, positive lymph node metastasis, high proliferative index, detectable p53 expression, advanced tumor stage, and high vascular invasion.

The level of an miRNA in a biological sample can be measured or determined using any technique that is suitable for detecting miRNA expression levels in a biological sample. Suitable techniques for determining miRNA expression levels in cells from a biological sample (e.g. Northern blot analysis, RT-PCR, quantitative RT-PCR, microRNA microarray, in situ hybridization, Next-generation sequencing (NGS) in the methods of the invention are well known to those of skill in the art. Alternatively, the level of an miRNA in a biological sample can be measured or determined indirectly by measuring abundance levels of cDNAs, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, or other molecules that are indicative of the expression level of the miRNA. Preferably, the level of an miRNA in a biological sample is determined indirectly in the methods of the invention by measuring abundance levels of cDNAs.

Accordingly, the present invention also contemplates an assay for use in a method of the invention and described above, comprising means and/or reagents for determining, directly or indirectly, the expression level of one or more miRNA associated with cancer stem cells in a biological sample from said subject. Preferably, the assay is based on microRNA microarray. Most preferably, the microRNA microarray comprises miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group comprising miR-363-3p, miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR-494.

The present invention also contemplates an miRNA inhibitor for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

An miRNA inhibitor is typically a nucleic acid molecule comprising and/or encoding an oligonucleotide with the reverse complement sequence of the miRNA it inhibits. The inhibitor hybridizes to the miRNA thereby negating or impairing its activity. The miRNA inhibitor typically comprises one or more modifications to enhance the hybridization of the inhibitor to the target miRNA. A nucleic acid chemical analog preferably comprises an RNA backbone, preferably with one or more modification such as a 2-0'-methyl modification, a locked nucleic acid (LNA) modification, a morpholino modification and/or a peptide nucleic acid (PNA) modification. The modifications can be at one or more sugar moieties of the backbone. Next to these chemical modifications, it has been shown that adding flanking sequences to the antisense-based miRNA inhibitors strongly enhances the inhibitory potency of these molecules. Preferably, these flanking

sequences form stem-loop hairpin structures with a total of between 8 and 16 bases on both 5' and 3' end of the antisense oligomer. Most preferred are flanking sequences of 12 bases, with a 4 base pair (2x4 bases) stem region and a loop region of 4 bases. These added structural elements are thought to enhance and stabilize the interaction between the miRNA-RISC complex and the inhibitor, thus prolonging the effect of the inhibitor. A preferred miRNA inhibitor of the invention is an miRNA inhibitor selected from the group comprising an siRNA directed against a mature miRNA, an shRNA directed against a mature miRNA, an shRNA or a siRNA capable of interfering the expression of short hairpin (sh), a nucleic acid or nucleic acid chemical analog capable of interfering with the activity or expression of one or more miRNA, a small RNA zipper and a morpholino antisense oligonucleotide capable of interfering with the expression of one or more miRNA. Preferably, the miRNA inhibitor of the invention is an oligonucleotide that interferes with the activity or expression of the miRNA of the invention by hybridizing to the nucleic acid sequence of the corresponding miRNA or to a variant or fragment thereof. Alternatively, the miRNA inhibitor is a single-stranded RNA molecule complementary to the specific miRNA of the invention, or to a variant or fragment thereof and that is conjugated to cholesterol.

Other examples of miRNA inhibitor are selected from the group comprising miRNA- sponge and miRNA mask (as described in 48-49).

As used herein, the term "variant" of an miRNA of the invention, includes a nucleic acid (e.g. RNA or DNA) substantially homologous to the original nucleic acid sequence of the miRNA, but which has at least one nucleotide different from that of the original sequence due to one or more deletions, insertions or substitutions. Substantially homologous means a variant nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the original miRNA nucleic acid sequence. A fragment of the nucleic acid sequence of an miRNA of the invention refers to a sequence substantially shorter than the original nucleic acid sequence of the miRNA. Typically, this fragment will have about 5 to 21 , in particular about 5, 6, 7, 8, 9 or 10 to 21 nucleotides, more particularly about 20 nucleotides e.g. 15-21 nucleotides, for example about 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the nucleic acid sequence of the corresponding miRNA.

An miRNA inhibitor of the invention as well as a variant or fragment thereof may be prepared by recombinant techniques using either prokaryotic or eukaryotic host cells. Alternatively, an miRNA inhibitor may be synthesized using commercially available synthesizers in known ways. Additional considerations can be taken into account when designing antisense oligonucleotides, including: (i) sufficient specificity in binding to the target sequence; (ii) solubility; (iii) stability against intra- and extracellular nucleases; (iv) ability to penetrate the cell membrane; and (v) when used to treat an organism, low toxicity. Preferably, the miRNA inhibitor of the invention is capable of interfering with the activity or expression of an miRNA selected from the group comprising miR-363-3p, miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR-494.

Most preferably, the miRNA inhibitor, variant or fragment thereof, is selected among the sequences comprising, or consisting in, the following sequences:

or a variant or fragment thereof. More preferably, the miRNA inhibitor of the invention is an miR-363-3p inhibitor, even more preferably an miR-363-3p inhibitor that comprises, or consists, in the

amino acid sequence set forth in SEQ ID NO: 1 (UACAGAUGGAUACCGUGCAAUU )or to a variant or fragment thereof. The present invention also contemplates an miRNA inhibitor in the form of a small-molecule that, preferably, regulates or interferes with the transcription of an miRNA of the invention. Also envisioned is a chemical agent, oligonucleotide or antibody that interferes with the expression of a target gene of the miRNA of the invention.

Also contemplated in the present invention is a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of one or more miRNA associated with cancer stem cells and a pharmaceutically acceptable carrier or diluent.

Usually, the pharmaceutical composition of the invention is for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

The term "therapeutically effective amount" as used herein means an amount of inhibitor of one or more miRNA associated with cancer stem cells high enough to significantly positively modify the symptoms and/or condition to be treated, but low enough to avoid serious side effects (at a reasonable risk/benefit ratio), within the scope of sound medical judgment. The therapeutically effective amount of the inhibitor of one or more miRNA associated with cancer stem cells is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the cancer.

"Pharmaceutically acceptable carrier or diluent" means a carrier or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes carriers or diluents that are acceptable for human

pharmaceutical use.

Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The pharmaceutical compositions may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling

materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include macrocrystalline cellulose, carboxymethyf cellulose sodium, polysorbate 80, phenyletbyl alcohol, chiorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S

PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991 ) which is incorporated by reference herein.

Further contemplated is a pharmaceutical composition comprising a

therapeutically effective amount of a host cell (e.g. single cell or population of cells) and a pharmaceutically acceptable carrier or diluent. Preferably, the host cell has been modified by the introduction of i) a gene delivery vector comprising one or more nucleic acid(s) encoding the miRNA inhibitor of the invention or ii) one or more nucleic acid(s) encoding the miRNA inhibitor of the invention. The pharmaceutical composition comprising a therapeutically effective amount of a host cell and a pharmaceutically acceptable carrier or diluent can be administered to the subject in need thereof by any method and route known in the art and described herein. The present invention further contemplates a method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising:

(b) administering said miRNA inhibitor to a subject suffering from breast cancer. Preferably, said miRNA inhibitor is in the form of a pharmaceutical composition comprising a therapeutically effective amount of said inhibitor as described above.

Alternatively, the method of treatment can be associated with a treatment selected from the group comprising radiotherapy, immunotherapy or hormone therapy. "Administering", as it applies in the present invention, refers to contact of an effective amount of an inhibitor of one or more miRNA of the invention, to the subject.

In general, methods of administering a composition comprising an miRNA inhibitor that are in the form of nucleic acids are well known in the art. In particular, the routes of administration already in use for nucleic acid therapeutics, along with formulations in current use, provide preferred routes of administration and formulation for the nucleic acids described herein.

Nucleic acid compositions can be administered by a number of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Nucleic acids can also be administered via liposomes or nanoparticules (as described in 44-47). Such administration routes and appropriate formulations are generally known to those of skill in the art.

Administration of the formulations (e.g. pharmaceutical compositions) described herein may be accomplished by any acceptable method which allows the miRNA inhibitor or nucleic acid encoding the miRNA inhibitor to reach its target. The particular mode selected will depend of course, upon factors such as the particular formulation, the severity of the state of the subject being treated, and the dosage required for therapeutic efficacy. The actual effective amounts of drug can vary according to the specific drug or combination thereof being utilized, the particular composition formulated, the mode of administration, and the age, weight, condition of the patient, and severity of the symptoms or condition being treated.

Any acceptable method known to one of ordinary skill in the art may be used to administer a formulation to the subject. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated.

Injections can be e.g., intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. The composition can be injected intradermally for treatment cancer, for example.

The injections can be given at multiple locations. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets.

Inhalation includes administering the composition with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the composition is encapsulated in liposomes.

Preferably, the agent and/or nucleic acid delivery system are provided in a manner which enables tissue-specific uptake of the agent and/or nucleic acid delivery system. Techniques include using tissue or organ localizing devices, such as wound dressings or transdermal delivery systems, using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capability and configured as expansive devices or stent grafts. The formulations may be delivered using a bio-erodible implant by way of diffusion or by degradation of the polymeric matrix.

The administration of the formulation may be designed so as to result in sequential exposures to the miRNA inhibitor over a certain time period, for example, hours, days, weeks, months or years. This may be accomplished, for example, by repeated administrations of a formulation or by a sustained or controlled release delivery system in which the miRNA inhibitor is delivered over a prolonged period without repeated administrations. Administration of the formulations using such a delivery system may be, for example, by oral dosage forms, bolus injections, transdermal patches or

subcutaneous implants. Maintaining a substantially constant concentration of the composition may be preferred in some cases.

Other delivery systems suitable include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones,

copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/or combinations of these. Microcapsules of the foregoing polymers containing nucleic acids are described in, for example, U.S. Patent No. 5,075,109. Other examples include nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems;

silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the miRNA inhibitor is contained in a formulation within a matrix (for example, as described in U.S. Patent Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), or diffusional systems in which an active component controls the release rate (for example, as described in U.S. Patent Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. The system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the miRNA inhibitor. In addition, a pump-based hardware delivery system may be used for delivery.

Also envisioned is a tumour-specific delivery of miRNA inhibitor-coupled superparamagnetic iron oxide nanoparticles as known in the art.

The present invention further contemplates a method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising modifying a host cell, and reintroducing the host cell (e.g. single cell or population of cells) into the patient in need thereof, i.e. suffering from breast cancer, breast cancer metastasis, or breast cancer drug resistance.

Preferably, a biopsy or other tissue or biological fluid sample comprising the single cell or the population of cells may be necessary. Cells such as fibroblast cells or stem cells that can be generated directly from adult cells, such as iPSCs, are particularly preferred in this regard. A gene delivery vector (e.g. plasmid or vector) or one or more nucleic acid(s) encoding the miRNA inhibitor can be introduced to host cell via one or more methods known in the art. These one or more methods include, without limitation, microinjection,

electroporation, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, optical transfection, proprietary agent- enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.

It will be appreciated that in the present method the modification following the

introduction of the gene delivery vector (plasmid or vector), or the one or more nucleic acid(s) encoding the miRNA inhibitor, to the host cell may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other aspects, it may occur in vivo.

The host cell(s) (e.g. single cell or population of cells) is then reintroduced into the patient in need thereof by any route of administration and/or delivery methods known in the art, as described herein.

Also envisioned is a method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising, comprising

i) determining the expression level of one or more miRNA associated with cancer stem cells in a biological sample obtained from a subject,

Preferably, the subject will be treated by a method of treatment selected from the group comprising surgery, radiotherapy, chemotherapy, immunotherapy or hormone therapy.

A chemotherapy of the present invention can concern as well agents that damage DNA and / or prevent cells from multiplying, such as genotoxins.

Genotoxins can be selected from the group comprising alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin,

mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil, cyclosphamide, iphosphamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazin, procarbazine, hexamefhyl melamine, triethylene melamine, busulfan, pipobroman, mitotane and other platine derivatives. An example of DNA cutters is bleomycin.

Topoisomerases poisons can be selected from the group comprising topotecan, irinotecan, camptothecin sodium salt, daorubicin, doxorubicin, idarubicin, mitoxantrone teniposide, adriamycin and etoposide.

Examples of DNA binders are dactinomycin and mithramycin whereas spindle poisons can be selected among the group comprising vinblastin, vincristin, navelbin, paclitaxel and docetaxel.

A chemotherapy of the present invention can concern as well antimetabolites selected among the following coumpounds: methotrexate, trimetrexate, pentostatin, cytarabin, ara- CMP, fludarabine phosphate, hydroxyurea, fluorouracyl, fioxuridine,

chlorodeoxyadenosine, gemcitabine, thioguanine and 6-mercaptopurine.

Radiotherapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy). External radiation therapy is most common and typically involves directing a beam of direct or indirect ionizing radiation to a tumor or cancer site. While the beams of radiation, the photons, the Cobalt or the particule therapy are focused to the tumor or cancer site, it is nearly impossible to avoid exposure of normal, healthy tissue. Energy source for external radiation therapy is selected from the group comprising direct or indirect ionizing radiation (for example: x-rays, gamma rays and particle beams or combination thereof).

Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, etc., inside the body, at, or near to the tumor site. Energy source for internal radiation therapy is selected from the group of radioactive isotopes

comprising: iodine (iodinel 25 or iodinel 31 ), strontium89, radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt, and combination thereof. Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, interstitial, and intracavity brachytherapy (high dose rate, low dose rate, pulsed dose rate). A currently less common form of internal radiation therapy involves biological carriers of radioisotopes, such as with radio-immunotherapy wherein tumor-specific antibodies bound to radioactive material are administered to a patient. The antibodies bind tumor antigens, thereby effectively administering a dose of radiation to the relevant tissue. Methods of administering radiation therapy are well known to those of skill in the art.

Also contemplated is a kit for performing a method according to the invention, said kit comprising a) means and/or reagents for determining the expression level of one or more miRNA associated with cancer stem cells a biological sample from said subject, and b) instructions for use.

For example, the kit may include reagents that specifically hybridize to one or more miRNA of the invention. Such reagents may be one or more nucleic acid molecule in a form suitable for detecting the one or more miRNA, for example, a probe or a primer. The kit may include reagents useful for performing an assay to detect one or more miRNAs, for example, reagents which may be used to detect one or more miRNAs in a qPCR reaction. The kit may likewise include a microarray useful for detecting one or more miRNAs

Probes and/or primers can be selected from those provided by the MicroRNA LNA™ PCR primer sets or specifically designed for detecting the one or more miRNA of the invention. The kit may further contain instructions for suitable operational parameters in the form of a label or product insert. For example, the instructions may include information or directions regarding how to collect a sample, how to determine the level of one or more miRNA in a sample, or how to correlate the level of one or more miRNA biomarkers in a sample with the status of a subject. The kit can contain one or more containers with miRNA biomarker samples, to be used as reference standards, suitable controls, or for calibration of an assay to detect the miRNA biomarkers in a test sample

Also contemplated is a kit comprising a composition or a pharmaceutical composition of the invention. The kit may further contain instructions that may include information or directions, drug quantity, composition, and so forth for the prescription.

The present invention also contemplates a gene delivery vector, preferably in the form of a plasmid or a vector, that comprises one or more nucleic acid(s) encoding an miRNA inhibitor of the present invention. As used herein, a "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).

Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g., E. coli plasmids col El, pCRI, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e. g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Various viral vectors are used for delivering nucleic acid to cells in vitro or in vivo. Non- limiting examples are vectors based on Herpes Viruses, Pox- viruses, Adeno-associated virus, Lentivirus, and others. In principle all of them are suited to deliver the expression cassette comprising an expressible nucleic acid molecule that codes for An miRNA inhibitor of the invention. In a preferred aspect, said viral vector is an adenoviral vector, preferably a replication competent adenovirus.

In a further aspect of the invention, a replication competent adenovirus according to the invention is a conditionally replicating adenovirus (CRAd). A CRAd will only replicate in cells in which the particular conditions exist that are required for replication of the CRAd.

A CRAd comprises an adenoviral genome from which one or more parts that are necessary for efficiently completing at least one step of the adenovirus infectious life cycle under certain physiological conditions (herein also "first conditions") but not under certain other physiological conditions (herein also "second conditions") have been modified, removed or have been otherwise engineered to be not expressed under the first conditions. Said first and second conditions could, e.g., be dictated by the

physiological conditions that exist in a particular type of cells (herein also "first cells"), but not in another type of cells (herein also "second cells"). Such a first type of cell is e.g. a cell derived from a particular type of tissue, where said cell contains a protein that is not or much less present in cells from other tissues (second type of cells). An example of a second type of cell is a cell that has lost proper cell growth control, such as e.g. a cancer cell, where said cell either lacks a protein that is present in cells that have not lost proper cell growth control or where said cell has gained expression (or over- expression) of a protein that is not or much less present in cells that have not lost proper cell growth control.

Another example of a second condition is a condition that exists in a particular stage of the cell cycle or in a particular developmental stage of the cell, where a certain protein is expressed specifically. Thus, CRAds can be designed such, that replication thereof is enabled in particular cells, such as cancer cells or a particular type of cancer cells, whereas in normal cells, replication of CRAds is not possible, or strongly reduced.

A preferred CRAd is provided by an adenovirus according to the invention, wherein said adenovirus comprises at least one mutation in one or more genes from the group consisting of E1A, E1 B, E4, and VA-RNAs, to achieve selective replication in tumors. An adenovirus according to the invention preferably carries a mutation in the E1A region encompassing at least a part of the CR2 domain of E1A, preferably a deletion

encompassing amino acids 122 to 129 (LTCHEAGF) of E1A. The term gene, as used herein, comprises the complete genomic region that is required for expression of a gene including, for example, the enhancer/promoter region and intronic en exonic sequences. An adenovirus according to the invention may further comprise modifications that increase its replication potential, such as e.g. overexpression of the E3-1 1 .6K ADP gene (Doronin et al., Virology, 305, 378-387, 2003) or deletion of the E1 B-19K gene (Sauthoff et al. Hum. Gene Ther. ll(2000):379-388), or that increase the replication selectivity for a certain type of cells, including but not limited to the modifications to make CRAds

(supra), or that reduce the immunogenicity (i.e., their potency to induce an immune response when introduced into an animal body), such as e.g. retention of the E3B region (Wang et al., Nature Biotechnol. 21 (2003):1328-1335).

An adenovirus according to the invention may further be modified to express one or more transgenes, such as e.g. a gene encoding a cytokine, a pro-apoptotic protein, an anti- angiogenic protein, a membrane fusogenic protein or a prodrug converting enzyme.

Expression control sequences for expression of an miRNA inhibitor in a target cell preferably comprise a polymerase II or polymerase III enhancer/promoter. A preferred polymerase II promoter for expression of a pri-miRNA is a selective RNA polymerase II promoter, such as a tissue- specific or a cell-specific promoter that directs expression of the miRNA inhibitor specifically or exclusively in the target cell. Expression control sequences for expression of an miRNA inhibitor preferably also comprise transcriptional stop sequences such as a poly(A) signal for polymerase ll-mediated expression, and a termination signal such as a stretch of at least 4 consecutive thymidine nucleotides for polymerase Ill-mediated expression. A preferred polymerase II promoter is selected from a CMV promoter, the immediate early gene of human cytomegalovirus, the SV40 promoter, and the long terminal repeat of Rous sarcoma virus. Another preferred promoter comprises regulatable elements, such as tetracycline, radiation or hormone regulated elements allowing control of the timing and level of transcription driven by the promoter. Preferred expression control sequences according to the invention comprise a selective RNA polymerase II promoter.

In another aspect, the one or more expression control sequences in an adenovirus according to the invention comprise an RNA polymerase III promoter. Preferred polymerase III promoter sequences are selected from the group consisting of 5S rRNA, tRNAs, VA RNAs, Alu RNAs, HI, and U6 small nuclear RNA promoter sequences.

The invention further provides a host cell (e.g. single cell or population of cells) that comprises a gene delivery vector (e.g. plasmid or vector) comprising an miRNA inhibitor of the present invention. Alternatively, the miRNA inhibitor of the invention is integrated in the host cells genomic DNA.

As used herein, the term "host cell" refers to a single cell or to a population of cells and may be a naturally occurring cell or a transformed cell that has been modified to comprise a vector of the invention and that supports the replication of the vector. The term includes cells into which an expression vector is initially introduced, and also to the progeny or potential progeny of such cells. Host cells may be cultured cells, explants, in vivo cells and the like.

Usually, the host cell is selected among the non-limiting group comprising, ES stem cells, pluripotent stem cells (such as iPSCs), epithelial cells, lymphocytes (such as Tumor-infiltrating lymphocytes (TILs)) and fibroblasts.

Also contemplated is a method of reducing a breast tumor size comprising:

(a) providing an inhibitor of one or more miRNA associated with cancer stem cells according to the invention ; and (b) contacting said miRNA inhibitor with a breast tumor.

Further contemplated is a method of preventing breast tumor growth or breast tumor cells growth comprising: (a) providing an inhibitor of one or more miRNA associated with cancer stem cells according to the invention; and (b) contacting said miRNA inhibitor with a breast tumor or with breast tumor cells.

The present invention also relates, in a particular aspect, to the use of miR-363- 3p as predictor of chemotherapy failure, using a simple blood-based test. Therefore, miR-363-3p is useful for cancer screening in early stage breast cancer to select patients at higher risk of recurrence and needing further specific therapies in addition to standard adjuvant treatments. Also contemplated is a nucleic acid encoding an miRNA, or an miRNA

inhibitor, of the invention, a fragment or variant thereof.

EXAMPLES

Example 1 Material & Methods

Cell lines

The following human breast cancer cell lines (MCF7, MDA-MB-231 , BT549, HCC70 and HCC38) were grown in the medium recommended by the ATCC. The normal-like breast cell line MCF10A were cultured in DMEM/F12 supplemented with 5% horse serum, EGF (100pg/nnl), hydrocortisone (0.48ng/ml), Insulin (4mg/ml) and Cholera Toxin (I mg/ml). All cell lines were maintained at 37°C in 5% CO2 humidified incubator. Human biological samples

Frozen human tumors and sera were obtained from 38 female patients with breast cancer enrolled in a neoadjuvant protocol (CHUV, Lausanne, protocol 30/10, Table 1 ). Biopsies as well as sera sampling were performed prior to and after the

chemotherapeutic treatments. Criteria for inclusion in the protocol were: locally advanced or inflammatory breast cancer, voluminous breast tumor with indication for neoadjuvant chemotherapy (cT4a, b, c, d, Nx or cTx, N2 or N3, or cT3cN0,1 or cT2cN0,1 ; according to the 7th TNM classification). The required age was over 18 years old. All patients approved and signed informed consent forms. The trial was conducted for 4 years and comprised 40 patients in total. This study relied on 38 patients for which a complete sample and dataset was available. Next-to-tumor samples were tissues surrounding breast tumors but considered as normal tissues and they were obtained from the frozen tissue bank of the Institute of pathology of

Lausanne, therefore they were independent of the previous described cohort. The trial was conducted in accordance with the Declaration of Helsinki, the Guidelines of Good Clinical Practice issued by ICH and Swiss regulatory authorities requirements. The trial was approved by the local ethical committee. Plasmid constructions and transfections

To modulate the level of miR363-3p in cells, the inventors used the BLOCK-iT™

Inducible Pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen, Thermofisher Scientific) allowing the regulated expression of miRNAs in mammalian cells under control of a tetracycline-regulated Pol II promoter. Pre-miRNA sequences for miR363- 3p and anti-miR363-3p were cloned into the destination vectors following the

manufacturer's instructions. Sequences are available in Table 3. The vector containing a miR-negative control provided by the manufacturer was used as control, and it was chosen to have no effect on gene regulation in mammalian cells.

For transfections, 150,000 MCF7 cells were seeded in 12-well plates. The day after plating, 1 pg of the vector of interest was transfected using 2μΙ of the JetPRIME reagent according to the manufacturer's instructions (Polyplus transfection SA, France). After three days, cells were recovered by trypsinization and seeded in a 10cm dish in the selection media. Resistant cells were selected by adding blasticidin at 10pg/nnl and G418 at 600pg/ml to the culture medium. The expression of miRNA was induced by the addition of 1 pg/ml of tetracycline to the culture media.

Mammosphere assay

Cells (4000 cells/cm2) were grown in ultralow attachment plates (Corning, USA) and in the MammoCult culture medium (Stemcells technologies, France). To generate chemoresistant mammospheres, cells were maintained in these conditions 3 days before a 48h-treatment with 5-fluorouracil (50mg/ml) or paclitaxel (100nM). Cells were then harvested for RNA extraction, or they were cultivated further during 5 days prior to sphere counting under light microscopy. Control and chemoresistant mammospheres were indistinguishable in terms of growth, size, or cell filling.

In vivo experiment

MCF7 cells stably transfected with miR-control or anti-miR363-3p were engineered to express luciferase through lentiviral infection with pCDH-CMV-MCS-EF1 -Puro-Luc2. These cells were then grown as mammospheres for three days as described in the previous section. Mammospheres were resuspended as single cells in DMEM 10% FCS at concentration of 10 000 cells/μΙ. 50 000 cells per gland were injected

intraductally via cleaved teat as previously described (24) but without surgically operating the mouse. NOD-SCID IL2Rgammanull used- mice were purchased from Jackson Laboratories and maintained and handled according to Swiss guidelines for animal safety in accordance with a protocol approved by the Swiss Institutional Animal Care and Use Committee. Mice were anesthetized by intra-peritoneal injection of 10 pL per g of 4,5 mg/kg Xylazin and 90 mg/kg Ketamin (Graeub AG). Immediately after injection mice were provided with doxycycline (2mg/ml, 2% sucrose in water) in the drinking water to induce the expression of constructs containing miRNAs. Tumor growth was monitored up to 45 days by bioluminescence detection with Xenogen MS Imaging System 200 (Caliper Life Sciences) in

accordance with the manufacturer's recommendations and protocols. Mice were euthanized and mammary glands, lungs, livers and brains were collected.

Anchorage independent Soft Agar colony

2500 cells per well were seeded in 6-well plates in DMEM medium supplemented with 0.35% agarose and 10% fetal bovine serum (FBS) with or without tetracycline as the inducer of miRNA expression. Cells were fed with complete medium every 3 days. After 21 days colonies were stained with 0.1 % cristal violet and counted.

Aldefluor assay and FACS sorting

The ALDEFLUOR™ fluorescent assay (Stemcell Technologies, France) allows the identification, evaluation, and isolation of stem and progenitor cells as based on the expression of aldehyde dehydrogenase (ALDH). Cells were grown under adherent conditions as monolayer and harvested as indicated above. After resuspension, the ALDH enzymatic reaction was assayed according to manufacturer's instructions, and cells were sorted using cytofluorometry. ALDH+ and ALDH- cell populations were processed for RNA extractions. RNA extraction and RT-qPCR

RNAs from cells or tissues were extracted using TRIzol (Life Technologies, Fisher Scientific) following the manufacturer's instructions, except that the volume of

isopropanol used at the precipitation step was increased from 500 μΙ to 800 μΙ to enhance the recovery of small RNAs. miRNAs were extracted from blood serum exosomes using miRCURYTM Exosome Isolation kit and miRCURYTM RNA

Isolation kit - biofluids (Exiqon, Denmark), following the manufacturer's

instructions. cDNA synthesis was performed using the miRCURY LNATM Universal RT microRNA PCR (Exiqon), providing templates for all microRNA, as for the real-time PCR assays. Real-time PCR were performed using SYBR green master mix (Roche, Basel, Switzerland) on a Light Cycler 480 instrument (Roche) using the following conditions: 45 amplification cycles at (95°C, 10s, 60°C, 1 min, ramp-rate 1 .6°C/s).

MicroRNA LNATM PCR primer sets from Exiqon were used to target hsa-miR-363-3p, U6 for normalization of miRNA levels in tissues and cells, and hsa-miR-16-5p for normalization of miRNA levels in the exosomes recovered from blood sera. miRNA microarray

The miRNA expression profiles of mammospheres were evaluated using the Agilent miRNA microarrays G4870A that bears 2006 probes for human miRNAs. Fluorescence was scanned with an Agilent G2566AA scanner and analyzed using the Feature

Extraction 10.7.3.1 software. Samples were quantile normalized as based on

summarized expression levels of probe sets. miRNAs that were differentially expressed when comparing MCF7- and MCF1 OA-derived mammospheres were identified using a moderated t-test implemented in the R Bioconductor limma package. P-values were adjusted across the two comparisons by the Benjamini-Hochberg method, controlling for false discovery rate. Log fold changes (M-value) between MCF7-mammospheres and chemoresistant MCF7-mammospheres were used to determine the chemoresistant miRNA signature.

Statistical Analysis

All quantified data represents the mean ± standard deviation of at least three samples. Statistical significance was determined by Student t test, or by Mann-Whitney and Wilcoxon tests for nonparametric populations. P values below 0.05 were considered as statistically significant.

Results

Identification of an miRNA chemoresistance signature in CSC-enriched cell populations.

In order to identify miRNAs involved in BCSC chemoresistance, MCF7 cancer cells were grown as mammospheres so as to enrich CSCs. The mammospheres were then further treated or not with 5-Fluorouracil (5-FU) or Paclitaxel (P), two chemotherapeutic agents used in BC therapy, to select for chemoresistant cells. RNAs were extracted from the resistant cells, or from untreated cells used as control, and they were profiled using microRNA microarrays. After normalization, 379 of the 2006 miRNAs present on the microarray were altered when comparing the untreated MCF7 mammospheres and those chemoresistant to both 5-FU and P (Figure 6). We observed 12 miRNAs specific to the MCF7 mammosphere cells that were resistant to 5-FU, while 5 miRNAs were specific to P-resistant MCF7 mammospheres. Among these miRNAs, 3 were similarly regulated in both the 5-FU- and the P-resistant mammospheres: miR-1290, miR-363-3p and miR-494 (Figure 1A).

In order to exclude miRNAs that are expressed in normal stem cells, we compared the miRNA profile of untreated mammospheres from MCF7 cells with those obtained from immortalized but non-tumorigenic MCF10A breast cells. When treated with 5-FU and P, all cells of the MCF10A mammospheres died, as may be expected from the lack of chemoresistant CSCs in the non-tumorigenic population. Overall, the levels of six miRNAs were altered in MCF7 mammospheres resistant to both 5-FU and P when compared to untreated non-tumorigenic MCF10A mammospheres (Figure 1 B). The miR-630 and miR-494 were downregulated, whereas miR-363-3p, miR-21 -3p, miR-142- 3p and miR-149-5p were overexpressed. Altogether, these miRNAs might define a breast cancer stem cells chemoresistance signature. To assess this possibility further, we focused our interest on miR363-3p, as it was the mostly highly overexpressed miRNA in the signature of both populations of chemoresistant mamnnospheres (Figure 6), whereas it had not been previously associated with BC.

High levels of miR-363-3p linked to chemoresistance and CSC marker expression.

The levels of miR-363-3p were quantified in CSC-enriched MCF7 cell populations selected or not for resistance to the 5-FU and P chemoreagents using RT-qPCR assays. This showed that the miRNA363-3p was 10-fold more expressed in CSC-enriched mammospheres when compared to adherent MCF7 cells (Figure 2A). It was also 12- and 60-fold overexpressed in mammospheres resistant to 5-FU and P, respectively (Figure 2A). The miR-363-3p was not expressed in MCF10A, whereas it was expressed in all breast cancer cell lines tested (Figure 7), indicating that the overexpression of this miRNA is specific marker of tumorigenic cell populations.

To further assess whether the expression of miRNA363-3p results from the presence of CSC in the cancer-derived cell populations, the ALDH stem cell and CSC marker was used to sort cells by cytofluorometry. ALDH+ and ALDH- populations sorted from the MCF7 and MDA- MB-231 tumorigenic cell lines were assessed for their levels of miR363-3p by RT-qPCR. A 20- and 100-fold increase in miR-363-3p levels was observed in the ALDH+ relative to the ALDH- cells, or when related to the whole MCF7 and MDA-MB-231 populations, respectively (Figure 2B). Overall, we concluded that miR363-3p is specifically expressed in BCSC, and even more specifically in CSC populations resistant to chemoreagents.

Effect of miRNA-363-3p on MCF7 mammosphere and colony formation in vitro.

To assess if the overexpression of miR363-3p may determine the CSC phenotype, we next attempted to up- or down-regulate its expression in MCF7 cells. MCF7 cell lines were generated to contain chromosome-integrated expression vectors consisting of co-cistronic coding sequences for An miRNA of interest and for the EmGFP gene (Emerald Green Fluorescent Protein), under the control of a tetracycline-inducible promoter. Three different constructs were transfected into MCF7, so as to mediate the ectopic overexpression of miR- 363-3p (MCF7-miR363-3p cells) or of miR-control, An miRNA negative control (MCF7-miR- control cells). Conversely, the action of the cellular miR-363-3p was inhibited by the expression of a complementary sequence (MCF7-anti- miR-363-3p cells).

When grown in adherent cell cultures, no significant difference of the miR-363-3p levels could be obtained in cells transfected with miR-control or with miR-363-3p (Figure 3A). Interestingly, a time course of the miR-363-3p levels revealed a transient increase following doxycycline addition, followed by a damped oscillation pattern towards the initial level, implying a homeostatic mechanism opposing the ectopic expression of this miRNA (data not shown). However transfection of the anti-miR-363-3p induced a stable two-fold decrease of the miR-363-3p levels. When the MCF7-anti-miR363-3p and MCF7-miR363-3p cells were grown as mammospheres, a decrease of the sphere size was observed upon miR-363-3p downregulation of (Figure 3B). Moreover, a 2-fold decrease in the number of spheres was observed when miR-363-3p was inhibited,, suggesting that high levels of miR-363-3p expression may be required to maintain the non-adherent growth ability typical of CSCs.

The possible role of miR-363-3p in tumorigenicity was further assessed using a colony soft agar assay performed with the tetracycline-inducible MCF7-anti-miR363-3p cells. After induction of the anti-miR expression by tetracycline, a significant downregulation of the numbers of colonies growing in soft agar conditions was observed, concomitant to a 4-fold decrease of the miR-363-3p levels (Figure 3C). Taken together, these results indicated that high levels of miR363-3p expression are required for efficient

mammosphere generation and colony formation in soft agar, taken as indicators of the CSC propagation and tumorigenicity of the MCF7 cell line.

Effect of miRNA-363-3p in MCF7 cells in vivo.

The evidence for the requirement of miR363-3p for CSC propagation in vitro, taken together with previously established links between CSCs and metastasis, prompted us to assess the effect of miR363-3p down-regulation on breast tumor growth in vivo, using an intraductal human-in-mouse transplantation model (24). Previous studies have shown that this model mimics well the natural microenvironment necessary for the malignant growth of human BC cells, providing an assay of intraductal tumor growth in vivo, as well as of the invasion into the stroma and the metastatisation process that may occur in late stages of tumor development.

In order to assess tumor growth in vivo, the MCF7-anti-miR-363-3p and MCF7-miR-c cell lines were transduced with an expression vector for the luciferase reporter. As the level of miR-363-3p were found to be higher in mammospheres than in adherent cells, we injected cells grown in mammosphere conditions, so as to enrich the populations in BCSC and precursor cells. After 3 days of culture in presence of doxycycline, the mammospheres were dissociated into single cell suspensions, and 50,000 cells were injected intraductally in the 4th and 5th murine mammary glands. The levels of miR-363- 3p at the time of injection were verified by RT-qPCR, showing a 4-fold decrease of miR- 363-3p expression in the MCF7- anti-miR-363-3p cells relative to those expressing the MCF7-miR-c control (Figure 4A, imbedded graph). Mice were provided doxycycline in the drinking water, so as to maintain expression of the anti-miR363-3p and control miR, and the growth of the tumors was followed by in vivo luciferase imaging of the live mice (Figure 4A). A significant delay in tumor growth was observed in the glands injected with the cells expressing the anti-miR363- 3p as compared to the controls. This indicated a role for miR-363-3p expression in rapid tumor growth in vivo.

After 6 weeks, the mice were sacrificed and the mammary glands were recovered, as well as lungs, livers and brains. Infiltration of MCF7 cells in the gland was assessed using GFP fluorescence imaging. Whole gland imaging indicated an increase in mammary ducts colonization in glands injected with MCF7-miR-c-expressing cells, as compared to those expressing anti-miR-363-3p (Figure 4B). Consistently, the human GAPDH was significantly less detected in glands of mice injected with MCF7-miR-363- 3pi compared to glands injected with MCF7-miRc, as assessed by RT-qPCR, providing a quantitative assessment of the proliferation and/or survival of the human tumor cells (Figure 4C). Moreover, no significant differences in EmGFP expression levels were found when normalized to the human GAPDH mRNA, which indicated that the

transcription of the miRNA expression cassette was maintained in vivo for both cell populations. Consistently, assays of the level of miR363-3p relative to human GAPDH revealed a significant decrease in the glands injected with the anti- miR-363-3p- expressing cells. Taken together, these results demonstrated that growth of MCF7 cells in the milk ducts was decreased upon the repression of miR363-3p.

In order to determine if miR363-3p may also be required for cell invasion in peripheral organs, we quantified the human GAPDH levels in lungs, livers and brains of mice.

These tissues were divided in several portions and GAPDH mRNA levels were determined for each part using RT-qPCR. Expression of human GAPDH was not detected in brains or livers of the two groups of mice. However, hGAPDH expression was readily detected in all area of the lungs of mice injected with MCF7-miRc cells, whereas only one sample of lung was positive for the mice injected with MCF7-anti- miR363-3p. The average of all assays revealed a statistically significant difference between the two groups (Figure 4D). Overall, these results showed that depletion of miR-363-3p expression impaired breast cancer stem cells establishment, growth and invasion in vivo.

Human BC biopsy miRNA-363-3p levels are linked to response to chemotherapies.

We next assayed the miR363-3p levels in biopsies of human tumor tissues obtained from 38 patients enrolled in a neoadjuvant chemotherapy protocol. Biopsies of the breast tumors and sera were obtained and assessed from the same patients before and after chemotherapy (Table 1 ). The pathologic Complete Response (pCR) of the responding patients was used as the study endpoint, and pCR was considered to be reached when no residual tumor could be found at the time of surgery, following chemotherapy. The patient's outcome was further recorded up to date during years following surgery.

The levels of miR-363-3p were assayed from biopsies obtained from each patient before and after chemotherapy, whereas a panel of 7 next-to-tumors tissues (NxT) was used as control. Hematoxylin Eosin (HE) staining was also performed for each sample, and respective percentages of tumor cells, immune cells and fibroblastic cells were determined by a pathologist (M.F.) in a blind study. The levels of miR-363-3p were significantly higher in tissues taken before chemotherapy relative to the biopsies obtained after chemotherapy and surgery, as well as relative to a next-to-tumor (NxT) tissues (Figure 5A).

Given that miRNA can be detected more easily in the exosomes released by tumor cells into the blood stream, the sera of patients were also collected before and after chemotherapy, from which exosomes were purified and processed for qPCR miRNA analysis. When assessing the seric levels of miR363-3p before and after chemotherapy, we observed either a decrease or an increase, depending on the patient (Figure 5B). Nevertheless, the miRNA-363-3p seric levels of patients with BC were found to be overall higher than those of two healthy donors, despite the treatment (data not shown). To analyze this further, we divided the BC patients into two subgroups (Table 2). The first subgroup comprised patients with low miR363-3p seric levels after chemotherapy, with a log2 fold change that was less than 6-fold higher than those of normal sera, whereas the second subgroup had high miR363-3p seric levels, with the log2 fold change over 6-fold vs. normal sera. Statistically highly significant difference was found when comparing the two subgroups, either before (Mann-Whitney test, p=0.029), or after

chemotherapy (Mann-Whitney test, p<0.0001 ). We then looked at the differences in miR363- 3p seric levels before or after chemotherapy within each subgroup. In the first subgroup, we observed a significant decrease of miR363-3p seric levels after

chemotherapy (Wilcoxon test, p=0.007), whereas a significant increase of miR363-3p seric levels was observed for the second subgroup after chemotherapy (Wilcoxon test, p=0.0005). This indicated that patients having low initial miR363-3p seric levels often displayed a further decrease upon chemotherapy, suggestive of a response to the treatment, whereas a rise upon chemotherapy was observed for patients that had higher initial seric levels and that did not respond to the treatment.

Analysis of the patients' pathologic and clinical reports indicated that patients from the second subgroup were mostly diagnosed with more aggressive breast tumors (Triple Negative or HER2 positive biology, 55%), whereas the majority of patients from first subgroup were classified with less aggressive luminal BC (60%, Table 2). Patients classified as having achieved a pCR at the time of surgery were observed in both subgroups, with 40 and 50% of pCR, respectively. However, when the patient outcome was analysed at later times, 4 had local or distant tumor relapse.

One patient, who had a local relapse, having refused a surgical intervention after chemotherapy, belonged to the first subgroup with low seric levels of miR363- 3p.

Interestingly, the level of miR-363-3p in the serum initially decreased after

chemotherapy, but then increased again, reaching the level observed at initial diagnosis, when the relapse was diagnosed. In contrast, the levels of miR494-3p and miR149-5p in the serum initially increased after chemotherapy, but then decreased again, reaching the level observed at initial diagnosis (Figure 8). According to this miRs signature, this patient responded well to the chemotherapy treatment. However, the lack of surgical intervention did not allow the removal of few remaining BC cells, which likely elicited the regrowth of the tumor and hence the relapse.

The three other patients, who displayed distal relapse and metastasis, belonged the second subgroup, as they had a high seric level of miR363-3p before chemotherapy, which remained elevated after chemotherapy, suggestive of resistance to

chemotherapy. Two of these three patients had passed away within the first year after breast cancer diagnosis. Overall, we concluded that initially high and further rising levels of miR363-3p correlated more often with a poor prognosis than the achievement of pCR.

Table 1 : Characteristics of 38 patients with breast cancer enrolled in a neoadjuvant chemotherapy protocol

Characteristics n (%)

Age (years)

mean (range) 47.4 (31-70)

Menopausal Status

Pre 25 (66)

Post 13 (34) ERa

Negative 17 (45)

Positive (>1% stained cells) 21 (55)

PR

Negative 29 (76)

Positive (> 1 % stained cells) 9 (24)

HER2

Negative 28 (74)

Positive 8 (21)

NA 2 (5)

Subtypes

Luminal (ER+, PR+and HER2-) 17 (45)

HER2+ 8 (21)

Triple Negative (ER-, PR-, HER-) 11 (29)

NA 2 (5)

Lymph Node Metastasis

Negative 25 (66)

Positive 11 (29)

NA 2 (5)

Histologic Grade

1 2 (5)

2 18 (47.5)

3 18 (47.5)

Pathologic Complete Response (pCR)

yes 16

no 22

ERa: Estrogen Receptor alpha; PR: Progesterone Receptor; HER2: Human Epidermal Growth Factor Receptor 2; NA: Non Available. Table 2: Classification of patients according to miR363-3p level variation in exosomes from sera before and after chemotherapy. miRNA-363-3p levels in exosomes from sera after chemotherapy

Log2 fold change to normal sera <6 >6

Range 4.2-6.0 6.2-17.8

Number of patient 15 22

Surrogate subtype of breast

cancer

LA / LB (%) 9 (60) 9 (41)

TN /HER2+ (%) 4 (27) 12 (55)

Unknown 2 1

pCR (%) 6 (40) 11 (50)

Follow up (years mean) 2.3 3.2

Number of relapsed patients

local 1 0

distal 0 3

LA: Luminal A (ER+, PR+, HER-); LB: Luminal B (ER+, PR+, HER+), TN: Triple negative (ER-, PR-, HER-); HER2+: Human Epidermal Growth Factor Receptor 2 positive (ER-, PR-, HER+); pCR: pathologic Complete Response. ^§Out of the 22 patients, only 17 had a follow up as 5 had unknown records.

Table 3: Summary of the sequences of oligonucleotides used for the construction of miRNA expression vectors.

Table 3.1 :

Table 4: miRNA sequences and Antisense synthetic sequences

References

1. Colleoni M, Viale G, Zahrieh D, Pruned G, Gentilini O, Veronesi P, et al. Chemotherapy is more effective in patients with breast cancer not expressing steroid hormone receptors: a study of preoperative treatment. Clin Cancer Res 2004;10(19):6622-8.

2. Fisher ER, Wang J, Bryant J, Fisher B, Mamounas E, Wolmark N. Pathobiology of preoperative chemotherapy: findings from the National Surgical Adjuvant Breast and Bowel (NSABP) protocol B-18. Cancer 2002;95(4):681-95.

3. Guarneri V, Broglio K, Kau SW, Cristofanilli M, Buzdar AU, Valero V, et al. Prognostic value of pathologic complete response after primary chemotherapy in relation to hormone receptor status and other factors. J Clin Oncol 2006;24(7): 1037-44.

4. Molyneux G, Regan J, Smalley MJ. Mammary stem cells and breast cancer. Cell Mol Life Sci 2007;64(24):3248-60.

5. Abdullah LN, Chow EK. Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med 2013;2(1):3.

6. Fillmore CM, Kuperwasser C. Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res 2008;10(2):R25.

7. O'Brien CS, Farnie G, Howell SJ, Clarke RB. Are stem-like cells responsible for resistance to therapy in breast cancer? Breast Dis 2008;29:83-9.

8. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100(7):3983-8.

9. Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K, et al. The CD44+/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res 2008;10(3):R53.

10. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003;17(10): 1253-70.

11. Farnie G, Clarke RB, Spence K, Pinnock N, Brennan K, Anderson NG, et al. Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst 2007;99(8):616-27.

12. Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero- Stanke L, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 2008;10(3):R52.

13. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005;65(13):5506-11.

14. Charafe-Jauffret E, Ginestier C, Iovino F, Tarpin C, Diebel M, Esterni B, et al. Aldehyde dehydrogenase 1 -positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res 2010;16(l):45-55.

15. Resetkova E, Reis-Filho JS, Jain RK, Mehta R, Thorat MA, Nakshatri H, et al. Prognostic impact of ALDH1 in breast cancer: a story of stem cells and tumor microenvironment. Breast Cancer Res Treat 2010;123(1):97-108.

16. Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, et al. Regulation by let-7 and lin- 4 miRNAs results in target mRNA degradation. Cell 2005;122(4):553-63.

17. Olsen PH, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN- 14 protein synthesis after the initiation of translation. Dev Biol 1999;216(2):671-80.

18. Mulrane L, McGee SF, Gallagher WM, O'Connor DP. miRNA dysregulation in breast cancer. Cancer Res 2013;73(22):6554-62.

19. Greene SB, Gunaratne PH, Hammond SM, Rosen JM. A putative role for microRNA- 205 in mammary epithelial cell progenitors. J Cell Sci 2010;123(Pt 4):606-18.

20. De Cola A, Volpe S, Budani MC, Ferracin M, Lattanzio R, Turdo A, et al. miR-205- 5p- mediated downregulation of ErbB/HER receptors in breast cancer stem cells results in targeted therapy resistance. Cell Death Dis 2015;6:el 823.

21. Ibarra I, Erlich Y, Muthuswamy SK, Sachidanandam R, Hannon GJ. A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells. Genes Dev 2007;21(24):3238-43.

22. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007;131(6): 1109-23.

23. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, et al. Downregulation of miRNA- 200c links breast cancer stem cells with normal stem cells. Cell 2009;138(3):592-603.

24. Behbod F, Kittrell FS, LaMarca H, Edwards D, Kerbawy S, Heestand JC, et al. An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ. Breast Cancer Res 2009;11(5):R66.

25. Hasegawa S, Eguchi H, Nagano H, Konno M, Tomimaru Y, Wada H, et al. MicroRNA- 1246 expression associated with CCNG2 -mediated chemoresistance and sternness in pancreatic cancer. Br J Cancer 2014;111 (8): 1572-80.

26. Bhardwaj A, Arora S, Prajapati VK, Singh S, Singh AP. Cancer "sternness"- regulating microRNAs: role, mechanisms and therapeutic potential. Curr Drug Targets 2013;14(10): 1175-84. 27. Neelakandan K, Babu P, Nair S. Emerging roles for modulation of microRNA signatures in cancer chemoprevention. Curr Cancer Drug Targets 2012;12(6):716-40.

28. Raza U, Zhang JD, Sahin O. MicroRNAs: master regulators of drug resistance, sternness, and metastasis. J Mol Med (Berl) 2014;92(4):321-36.

29. Wang J, Yang M, Li Y, Han B. The Role of MicroRNAs in the Chemoresistance of Breast Cancer. Drug Dev Res 2015;76(7):368-74.

30. Sun X, Jiao X, Pestell TG, Fan C, Qin S, Mirabelli E, et al. MicroRNAs and cancer stem cells: the sword and the shield. Oncogene 2014;33(42):4967-77.

31. Pfeffer SR, Yang CH, Pfeffer LM. The Role of miR-21 in Cancer. Drug Dev Res 2015.

32. Sicard F, Gayral M, Lulka H, Buscail L, Cordelier P. Targeting miR-21 for the therapy of pancreatic cancer. Mol Ther 2013;21(5):986-94.

33. Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA 2008;14(11):2348-60.

34. Yan LX, Wu QN, Zhang Y, Li YY, Liao DZ, Hou JH, et al. Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth. Breast Cancer Res 2011;13(1):R2.

35. Chan JK, Blansit K, Kiet T, Sherman A, Wong G, Earle C, et al. The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer. Gynecol Oncol 2014;132(3):739-44.

36. Warnecke-Eberz U, Chon SH, Holscher AH, Drebber U, Bollschweiler E. Exosomal onco- miRs from serum of patients with adenocarcinoma of the esophagus: comparison of miRNA profiles of exosomes and matching tumor. Tumour Biol 2015;36(6):4643-53.

37. Li XT, Wang HZ, Wu ZW, Yang TQ, Zhao ZH, Chen GL, et al. miR-494-3p Regulates Cellular Proliferation, Invasion, Migration, and Apoptosis by PTEN/AKT Signaling in Human Glioblastoma Cells. Cell Mol Neurobiol 2015;35(5):679-87.

38. Xu S, Wei J, Wang F, Kong LY, Ling XY, Nduom E, et al. Effect of miR-142-3p on the M2 macrophage and therapeutic efficacy against murine glioblastoma. J Natl Cancer Inst 2014;106(8). 39. Cao JX, Lu Y, Qi JJ, An GS, Mao ZB, Jia HT, et al. MiR-630 inhibits proliferation by targeting CDC7 kinase, but maintains the apoptotic balance by targeting multiple modulators in human lung cancer A549 cells. Cell Death Dis 2014;5:el426.

40. Corcoran C, Rani S, Breslin S, Gogarty M, Ghobrial IM, Crown J, et al. miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in

HER2 over-expressing breast cancer. Mol Cancer 2014;13:71.

41. Hsu KW, Wang AM, Ping YH, Huang KH, Huang TT, Lee HC, et al. Downregulation of tumor suppressor MBP-1 by microRNA-363 in gastric carcinogenesis. Carcinogenesis 2014;35(1):208-17.

42. Han H, Sun D, Li W, Shen H, Zhu Y, Li C, et al. A c-Myc-MicroRNA functional feedback loop affects hepatocarcinogenesis. Hepatology 2013;57(6):2378-89.

43. Ng SB, Yan J, Huang G, Selvarajan V, Tay JL, Lin B, et al. Dysregulated microRNAs affect pathways and targets of biologic relevance in nasal-type natural killer/T-cell lymphoma. Blood 2011 ;118(18):4919-29.

44. Fan W, Wang X, Ding B, Cai H, Wang X, Fan Y, et al. Thioaptamer-conjugated CD44-targeted delivery system for the treatment of breast cancer in vitro and in vivo. J Drug Target 2015: 1-13.

45. Kang L, Gao Z, Huang W, Jin M, Wang Q. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment. Acta Pharm Sin B 2015;5(3): 169- 75.

46. Kheirolomoom A, Kim CW, Seo JW, Kumar S, Son DJ, Gagnon MK, et al. Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE(-/-) Mice. ACS Nano 2015;9(9):8885-97.

47. Shu D, Li H, Shu Y, Xiong G, Carson WE, 3rd, Haque F, et al. Systemic Delivery of Anti- miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology. ACS

Nano 2015;9 (10):9731-40.

48. Ebert M. S., Neilson J. R. & Sharp P. A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4, 721-726 (2007).

49. Wang Z. The principles of MiRNA-masking antisense oligonucleotides technology. Methods Mol. Biol. 676, 43-49 (2011).

Claims

1 . A method for diagnosing a breast cancer in a subject and for predicting the resistance to chemotherapy in said subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological sample from said subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of the subject having or developing resistance to chemotherapy.

2. The method of claim 1 , wherein the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-5p, miR-149-5p, miR-630 and miR-494.

3. The method of claim 1 or 2, wherein the alteration in the level of one or more miRNA corresponds to an upregulated or a downregulated expression of said one or more miRNA.

4. The method of any one of the preceding claims, wherein the

upregulated expression of one or more miRNA in a biological sample corresponds to an increase equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer- free subject.

5. The method of any one of claims 1 to 4, wherein the downregulated expression of one or more miRNA in a biological sample corresponds to a diminution equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject.

6. The method of any one of the preceding claims, wherein the one or more miRNA associated with cancer stem cells is miR-363-3p.

7. The method of any one of claims 1 to 4 and 7, wherein an upregulated expression of miR-363-3p is indicative of the subject having or developing resistance to chemotherapy.

8. The method of any one of the preceding claims, wherein the

chemotherapy is neoadjuvant chemotherapy.

9. The method of any one of the preceding claims, wherein the

chemotherapy is selected from the group of drugs comprising doxorubicin,

carboplatin, cyclophosphamide, epirubicin, fluorouracil (5-FU), methotrexate, paclitaxel, docetaxel, or a combination of one or more of these drugs.

10. The method of any one of the preceding claims, wherein the biological sample is selected from the group comprising whole blood, serum, serum exosomes, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, and cancer cells or a combination of one or more of these biological samples.

1 1 . A method for the diagnosis of metastatic cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

12. A method for predicting the metastatic ability of a cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

13. A method for determining the progression or regression of cancer in a subject suffering from breast cancer, said method comprising

(a) providing a biological sample from said subject,

14. A method of predicting the likelihood of metastasis-free survival of a breast cancer patient, said method comprising

(a) providing a biological sample from said subject,

wherein an alteration in the level of one or more miRNA associated with cancer stem cells in said biological subject, relative to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject, is indicative of metastasis-free survival of a breast cancer patient.

15. The method of any one of claim 1 1 -14, wherein the one or more miRNA associated with cancer stem cells is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-5p, miR-149-5p, miR-630 and miR-494.

16. The method of any one of claims 1 1 -15, wherein the alteration in the level of one or more miRNA corresponds to an upregulated or a downregulated expression of said one or more miRNA.

17. The method of any one of claims 1 1 -16, wherein the upregulated expression of one or more miRNA in a biological sample corresponds to an increase equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject.

18. The method of any one of claims 1 1 -16, wherein the downregulated expression of one or more miRNA in a biological sample corresponds to a diminution equal or superior to 5 %, preferably equal or superior to 20 %, more preferably equal or superior to 40 %, most preferably equal or superior to 60 %, more preferably equal or superior to 500%, even more preferably equal or superior to 1000 %, in particular equal or superior to 5000 % when compared to the level of corresponding said one or more miRNA in a control biological sample of a cancer-free subject.

19. The method of any one of claims 1 1 to 18, wherein the one or more miRNA associated with cancer stem cells is miR-363-3p.

20. The method of any one of claims 1 1 -19, wherein the biological sample is selected from the group comprising whole blood, serum, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, and cancer cells or a combination of one or more of these biological samples.

21 . An assay for use in a method of anyone of the preceding claims, comprising means and/or reagents for determining the expression level of one or more miRNA associated with cancer stem cells in a biological sample from said subject.

22. The assay of claim 21 , wherein the assay is based on microRNA microarray.

23. The assay of claim 22, wherein it comprises miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group comprising miR- 363-3p, miR-21 -3p, miR142-3p, miR-149-5p, miR-630 and miR-494.

24. An miRNA inhibitor for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

25. The miRNA inhibitor of claim 24, wherein it is selected from the group comprising an siRNA directed against a mature miRNA, an shRNA directed against a mature miRNA, an shRNA or a siRNA capable of interfering the expression of short hairpin (sh), a nucleic acid or nucleic acid chemical analog capable of interfering with the activity or expression of one or more miRNA, and a morpholino antisense oligonucleotide capable of interfering with the expression of one or more miRNA.

26. The miRNA inhibitor of claim 24 or 25, wherein the breast cancer, the breast cancer metastasis, or the breast cancer drug resistance is a cancer stem cell breast cancer.

27. The miRNA inhibitor of any one of claims 24-26, wherein the miRNA inhibitor is an oligonucleotide that interferes with the activity or expression of the miRNA by hybridizing to the nucleic acid sequence of the corresponding miRNA or to a variant or fragment thereof.

28. The miRNA inhibitor of any one of claims 24-27, wherein the one or more miRNA is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-5p, miR-149-5p, miR-630 and miR-494.

29. The miRNA inhibitor of any one of claims 24-28, wherein the variant of a nucleic acid miRNA includes a nucleic acid substantially homologous to the original nucleic acid sequence of the miRNA.

30. The miRNA inhibitor of any one of claims 24-29, wherein the miRNA inhibitor is selected among the sequences comprising, or consisting in, the following sequences:

or to a variant or fragment thereof.

31 . A method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising:

32. The method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance of claim 31 , comprising:

(a) providing an inhibitor of one or more miRNA associated with cancer stem cells of anyone of claims 24-30; and

33. A pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of one or more miRNA associated with cancer stem cells and a pharmaceutically acceptable carrier or diluent.

34. The pharmaceutical composition of claim 33 comprising a

therapeutically effective amount of an inhibitor of anyone of claims 24-30.

35. The pharmaceutical composition of claims 33 or 34, wherein the one or more miRNA is selected from the group comprising miR-363-3p, miR-21 -3p, miR142-5p, miR-149-5p, miR-630 and miR-494.

36. A composition comprising an inhibitor of one or more miRNA of anyone of claims 24-30.

37. A pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of one or more miRNA of anyone of claims 24-30 for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.

38. A kit for performing a method according to any one of claims 1 to 20, said kit comprising

a) means and/or reagents for determining the expression level of one or more miRNA associated with cancer stem cells a biological sample from said subject, and b) instructions for use.

39. A method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising, comprising

40. A method of reducing a breast tumor size comprising:

(a) providing an inhibitor of one or more miRNA associated with cancer stem cells of anyone of claims 24-30 ; and

(b) contacting said miRNA inhibitor with a breast tumor.

41 . A method of preventing breast tumor growth or breast tumor cells growth comprising:

(b) contacting said miRNA inhibitor with a breast tumor or with breast tumor cells.

42. A plasmid or a vector comprising one or more nucleic acid(s) encoding the miRNA inhibitor of anyone of claims 24-30.

43. A host cell comprising a plasmid or vector of claim 42 or one or more nucleic acid(s) encoding the miRNA inhibitor of anyone of claims 24-30.

44. A pharmaceutical composition comprising a therapeutically effective amount of a host cell of claim 43 and a pharmaceutically acceptable carrier or diluent.

45. A method of treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance comprising

(a) providing a host cell of claim 43 or a pharmaceutical composition of claim 44, and (b) introducing or reintroducing the host cell or the pharmaceutical composition into the patient in need thereof.

46. A nucleic acid encoding an miRNA inhibitor of anyone of claims 24-30.