WO2017207623A1 - Miarn utilisés en tant que biomarqueurs et régulateurs de cellules souches cancéreuses - Google Patents

Miarn utilisés en tant que biomarqueurs et régulateurs de cellules souches cancéreuses Download PDF

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WO2017207623A1
WO2017207623A1 PCT/EP2017/063143 EP2017063143W WO2017207623A1 WO 2017207623 A1 WO2017207623 A1 WO 2017207623A1 EP 2017063143 W EP2017063143 W EP 2017063143W WO 2017207623 A1 WO2017207623 A1 WO 2017207623A1
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mirna
cancer
mir
breast cancer
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Stéphanie Renaud
Nicolas Mermod
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Université de Lausanne
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/10—Production naturally occurring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • 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.
  • BC Breast cancer
  • neoadjuvant chemotherapy initially reserved to treat advanced and inflammatory BC before surgical intervention, is not always beneficial (1 -3).
  • additional biomarkers are needed to better predict the failure of neoadjuvant systemic therapies and BC recurrence.
  • CSCs Cancer Stem Cells
  • CSCs are thought to be capable of both self-renewal and of differentiation to other cancer cell types.
  • 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.
  • BCSCs breast cancer stem cells
  • miRNA 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).
  • 3'-UTR 3'-untranslated region
  • miRNA dysregulation has been shown to impact cancer occurrence and resistance to treatment (18).
  • 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 ).
  • 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 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 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
  • 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 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 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 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 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,
  • FIG. 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.
  • FIG. 1 miRNA-363-3p expression in breast cancer cell lines.
  • FIG. 3 Effect of miRNA-363-3p inhibition in MCF7 cell line.
  • B The effect of miRNA-363-3p inhibition on mammospheres size and number was determined by fluorescence microscopy.
  • FIG. 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
  • mice mammary glands intraductally in mice mammary glands, as indicated, and mice were provided with doxycycline in the drinking water.
  • 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).
  • FIG. 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 ).
  • 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.
  • 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.
  • FIG. 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).
  • 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.
  • these miRNAs can be, e.g. used to constitute a signature associated with the occurrence of the disease and its response to chemotherapy.
  • 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.
  • the subject is a subject in need of treatment or a subject suffering from breast cancer.
  • 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.
  • treatment 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:
  • the disease is breast cancer, breast cancer metastasis, or breast cancer drug resistance.
  • 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;
  • 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;
  • Triple-negative/basal-like breast cancer is hormone-receptor negative
  • 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.
  • 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 stem cells have 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.
  • CSCs cancer stem cells
  • chemoresistance 26-30
  • miRNAs 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.
  • nucleic acid refers 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.
  • oligonucleotides or nucleotides can comprise
  • nucleobases 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-
  • 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); cyclohe
  • 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.
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • 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.
  • the inventors 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • miRNA-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.
  • miR-363-3p 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.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • miR-363-3p, miR-21 -3p, miR142-3p and miR-149-5p are overexpressed in chemoresistant BCSC, whereas miR-630 and miR494 are downregulated.
  • an upregulated expression of miR-363-3p is indicative of the subject having or developing resistance to chemotherapy.
  • the chemotherapy is adjuvant or neoadjuvant chemotherapy
  • 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.
  • 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.
  • FEC which is a combination of 5-FU, epirubicin and cyclophosphamide
  • TAC which is a combination of docetaxel, doxorubicin and cyclophosphamide.
  • 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.
  • FSA fine needle aspiration
  • core-needle biopsy core-needle biopsy
  • excisional biopsy incisional biopsy
  • incisional biopsy incisional biopsy
  • punch biopsy punch biopsy.
  • the invention further contemplates a method for the diagnosis of metastatic cancer in a subject suffering from breast cancer, said method comprising
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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”).
  • 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 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.
  • 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”).
  • 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the level of an miRNA in a biological sample is determined indirectly in the methods of the invention by measuring abundance levels of cDNAs.
  • 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.
  • the assay is based on microRNA microarray.
  • 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.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • 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.
  • 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.
  • 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.
  • miRNA inhibitor are selected from the group comprising miRNA- sponge and miRNA mask (as described in 48-49).
  • 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.
  • 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.
  • 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.
  • 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.
  • the miRNA inhibitor, variant or fragment thereof is selected among the sequences comprising, or consisting in, the following sequences:
  • 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
  • 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.
  • 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.
  • the pharmaceutical composition of the invention is for use in the treatment of breast cancer, breast cancer metastasis, or breast cancer drug resistance.
  • therapeutically effective amount 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
  • 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.
  • compositions include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • 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
  • compositions 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.
  • 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.
  • composition comprising a
  • a host cell e.g. single cell or population of cells
  • a pharmaceutically acceptable carrier or diluent e.g. a pharmaceutically acceptable carrier or diluent.
  • 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:
  • said miRNA inhibitor is in the form of a pharmaceutical composition comprising a therapeutically effective amount of said inhibitor as described above.
  • the method of treatment can be associated with a treatment selected from the group comprising radiotherapy, immunotherapy or hormone therapy.
  • administering refers to contact of an effective amount of an inhibitor of one or more miRNA of the invention, to the subject.
  • nucleic acid therapeutics 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.
  • 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.
  • 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.
  • 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.
  • an aerosol in an inhaler
  • 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
  • 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.
  • 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,
  • 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.
  • a pump-based hardware delivery system may be used for delivery.
  • 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.
  • a host cell e.g. single cell or population of cells
  • 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
  • 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,
  • 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
  • the host cell(s) 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
  • 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.
  • alkylating agents are lomustine, carmustine, streptozocin,
  • 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.
  • 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,
  • Radiotherapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells.
  • 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
  • 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.
  • kits for performing a method according to the invention 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.
  • 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 LNATM 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.
  • 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
  • kits 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.
  • 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.
  • yeast plasmids such as the 2 ⁇ 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.
  • 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.
  • said viral vector is an adenoviral vector, preferably a replication competent adenovirus.
  • a replication competent adenovirus according to the invention is a conditionally replicating adenovirus (CRAd).
  • CRAd conditionally replicating adenovirus
  • 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.
  • first and second conditions could, e.g., be dictated by the
  • first cells physiological conditions that exist in a particular type of cells
  • 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).
  • 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).
  • second type of cells 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.
  • 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
  • gene 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
  • 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.
  • 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.
  • 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.
  • a gene delivery vector e.g. plasmid or vector
  • the miRNA inhibitor of the invention is integrated in the host cells genomic DNA.
  • 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.
  • 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.
  • ES stem cells pluripotent stem cells (such as iPSCs), epithelial cells, lymphocytes (such as Tumor-infiltrating lymphocytes (TILs)) and fibroblasts.
  • pluripotent stem cells such as iPSCs
  • epithelial cells such as lymphocytes (such as Tumor-infiltrating lymphocytes (TILs))
  • TILs Tumor-infiltrating lymphocytes
  • Also contemplated is a method of reducing a breast tumor size comprising:
  • 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
  • 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.
  • EGF 100pg/nnl
  • hydrocortisone 0.48ng/ml
  • Insulin 4mg/ml
  • Cholera Toxin I mg/ml
  • 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 neoadjuvant protocol (CHUV, Lausanne, protocol 30/10, Table 1 ). Biopsies as well as sera sampling were performed prior to and after the neoadjuvant protocol (CHUV, Lausanne, protocol 30/10, Table 1 ). Biopsies as well as sera sampling were performed prior to and after the CHUV, Lausanne, protocol 30/10, Table 1 ).
  • chemotherapeutic treatments include: 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
  • 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.
  • 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/ ⁇ . 50 000 cells per gland were injected
  • 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
  • mice were euthanized and mammary glands, lungs, livers and brains were collected.
  • the ALDEFLUORTM fluorescent assay allows the identification, evaluation, and isolation of stem and progenitor cells as based on the expression of aldehyde dehydrogenase (ALDH).
  • ALDEFLUORTM 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).
  • ALDEFLUORTM fluorescent assay Stemcell Technologies, France
  • ALDH aldehyde dehydrogenase
  • RNAs from cells or tissues were extracted using TRIzol (Life Technologies, Fisher Scientific) following the manufacturer's instructions, except that the volume of
  • miRNAs were extracted from blood serum exosomes using miRCURYTM Exosome Isolation kit and miRCURYTM RNA
  • 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
  • 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.
  • 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).
  • miR-630 and miR-494 were downregulated, whereas miR-363-3p, miR-21 -3p, miR-142- 3p and miR-149-5p were overexpressed.
  • miR-363-3p, miR-21 -3p, miR-142- 3p and miR-149-5p were overexpressed.
  • miR363-3p 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.
  • 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.
  • 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).
  • miR363-3p is specifically expressed in BCSC, and even more specifically in CSC populations resistant to chemoreagents.
  • 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.
  • MCF7-miR363-3p cells 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).
  • MCF7-miR363-3p cells An miRNA negative control
  • the MCF7-anti-miR-363-3p and MCF7-miR-c cell lines were transduced with an expression vector for the luciferase reporter.
  • miR-363-3p were found to be higher in mammospheres than in adherent cells, 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.
  • 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).
  • 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).
  • no significant differences in EmGFP expression levels were found when normalized to the human GAPDH mRNA, which indicated that the
  • 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).
  • 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.
  • Table 1 Characteristics of 38 patients with breast cancer enrolled in a neoadjuvant chemotherapy protocol
  • 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
  • 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.
  • RNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis.

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Abstract

La présente invention concerne des méthodes et des compositions pharmaceutiques pour le traitement du cancer du sein, et pour le diagnostic du cancer du sein, de sa métastase et de la résistance aux médicaments du cancer du sein.
PCT/EP2017/063143 2016-05-31 2017-05-31 Miarn utilisés en tant que biomarqueurs et régulateurs de cellules souches cancéreuses WO2017207623A1 (fr)

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WO2021182881A1 (fr) * 2020-03-12 2021-09-16 (주) 바이오인프라생명과학 Multiples biomarqueurs pour le diagnostic du cancer du sein et utilisation associée
CN113913524A (zh) * 2021-11-09 2022-01-11 上海市第十人民医院 早期乳腺癌诊断模型及诊断系统
US11236337B2 (en) 2016-11-01 2022-02-01 The Research Foundation For The State University Of New York 5-halouracil-modified microRNAs and their use in the treatment of cancer

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US11236337B2 (en) 2016-11-01 2022-02-01 The Research Foundation For The State University Of New York 5-halouracil-modified microRNAs and their use in the treatment of cancer
CN110184351A (zh) * 2019-05-29 2019-08-30 南通普惠精准医疗科技有限公司 检测试剂盒的应用
WO2021182881A1 (fr) * 2020-03-12 2021-09-16 (주) 바이오인프라생명과학 Multiples biomarqueurs pour le diagnostic du cancer du sein et utilisation associée
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KR102414106B1 (ko) 2020-03-12 2022-06-29 (주) 바이오인프라생명과학 유방암 진단용 다중 바이오마커 및 이의 용도
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