WO2016140552A1 - Composition de biomarqueur pour le diagnostic de la sensibilité à un agent anticancéreux dans le cancer du sein résistant à un agent anticancéreux - Google Patents

Composition de biomarqueur pour le diagnostic de la sensibilité à un agent anticancéreux dans le cancer du sein résistant à un agent anticancéreux Download PDF

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WO2016140552A1
WO2016140552A1 PCT/KR2016/002215 KR2016002215W WO2016140552A1 WO 2016140552 A1 WO2016140552 A1 WO 2016140552A1 KR 2016002215 W KR2016002215 W KR 2016002215W WO 2016140552 A1 WO2016140552 A1 WO 2016140552A1
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mir
anticancer
ly6k
breast cancer
expression
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박종훈
공현경
김예솔
이연선
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숙명여자대학교산학협력단
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Priority claimed from KR1020150113745A external-priority patent/KR101581721B1/ko
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Definitions

  • the present invention relates to a biomarker composition for diagnosing anticancer sensitivity of an anticancer drug-resistant breast cancer comprising LY6K or miR-192-5p, a diagnostic kit, a diagnostic method, and a pharmaceutical composition for enhancing anticancer effect comprising a LY6K expression inhibitor or a miR-192-5p inhibitor.
  • a biomarker composition for diagnosing anticancer sensitivity of an anticancer drug-resistant breast cancer comprising LY6K or miR-192-5p
  • a diagnostic kit a diagnostic method
  • a pharmaceutical composition for enhancing anticancer effect comprising a LY6K expression inhibitor or a miR-192-5p inhibitor.
  • the mechanism of resistance to anti-hormonal drugs such as tamoxifen, a breast cancer drug, is revealed, and it was confirmed that miR-192-5p inhibition in LY6K overexpressing breast cancer cells restored tamoxifen sensitivity by ER ⁇ re-expression.
  • the present invention is an anti-cancer drug sensitivity diagnostic biomarker composition, diagnostic kit, diagnostic method of the anti-cancer drug-resistant breast cancer comprising miR-500a-3p and a pharmaceutical composition for enhancing anticancer effect comprising any one of miR-500a-3p or its expression promoter.
  • the mechanism of resistance to anti-hormonal drugs such as tamoxifen, a breast cancer drug, is disclosed, and the activation of miR-500a-3p in LY6K overexpressing breast cancer cells has recovered tamoxifen sensitivity by ER ⁇ re-expression. And inhibited sensitivity to anticancer drugs.
  • breast cancer is a common cancer in women around the world. According to practice breast cancer is divided into several subgroups depending on the expression of receptors such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Most breast cancers are ER dependent. However, about 30% of breast cancers are classified as ER negative breast cancers, especially ER ⁇ negative breast cancers. ER ⁇ negative breast cancers do not respond to anti-estrogen therapy and tamoxifen, a selective estrogen receptor modulator.
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • Tamoxifen is clinically associated with growth inhibition and apoptosis in breast cancer cells by disturbing estrogen binding to ER in ER ⁇ positive breast cancers, but it is well known that apoptosis occurs only at high tamoxifen concentrations in ER ⁇ negative breast cancers.
  • Lymphocyte antigen 6 complex locus K is a Ly-6 / urokinase-type plasminogen activator receptor (uPAR) superfamily member.
  • LY6K has been reported as a molecular biomarker of esophageal squamous cell carcinomas, bladder cancer and breast cancer. In breast cancer cells, LY6K expression is mediated by AP-1 transcription factors and enhances cell proliferation, invasion and metastatic capacity. However, little is known about the effect of LY6K on breast cancer.
  • MicroRNA is a small non-coding RNA consisting of 20-22 nucleotides. miRNAs recognize seed sequences of several target genes by binding to the 3'UTR and then degrade or inhibit translation of the target mRNA. In many carcinomas, various cellular activities such as metabolism, apoptosis and proliferation are regulated by miRNAs. The relevance of miRNAs for increasing tamoxifen sensitivity has been studied. For example, re-expression of miR-320a, miR-375 and miR-342 can restore tamoxifen sensitivity by inhibiting the target.
  • An object of the present invention is to provide a biomarker composition, a diagnostic kit, a diagnostic method, a pharmaceutical composition for enhancing anti-cancer effects, or a cancer composition for preventing or treating cancer.
  • the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, including LY6K.
  • the present invention provides an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer, comprising an agent for detecting LY6K.
  • the present invention comprises the steps of analyzing the expression of LY6K in a sample isolated from the subject; And comparing the expression level with the LY6K expression level in the normal group, providing a method for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer.
  • the present invention provides a pharmaceutical composition for enhancing the anticancer effect comprising a LY6K inhibitor and enhances the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.
  • the present invention includes a LY6K inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer agent resistant breast cancer.
  • the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer drug resistant breast cancer, comprising miR-192-5p.
  • an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer comprising an agent for detecting miR-192-5p.
  • the present invention comprises the steps of analyzing miR-192-5p expression in a sample isolated from the individual; And comparing the expression level with miR-192-5p expression level in the normal group, and provides an anticancer drug sensitivity diagnosis method for anticancer drug resistant breast cancer.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer disease comprising a miR-192-5p inhibitor and enhancing the anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.
  • the present invention includes a miR-192-5p inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating a cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
  • the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, comprising miR-500a-3p.
  • the present invention provides an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-500a-3p.
  • the present invention comprises the steps of analyzing miR-500a-3p expression in a sample isolated from the individual; And comparing the expression level with miR-500a-3p expression levels in the normal group, and provides an anti-cancer drug sensitivity diagnostic method for anti-cancer drug-resistant breast cancer.
  • the present invention provides a pharmaceutical composition for enhancing the anticancer effect comprising miR-500a-3p or an expression promoter thereof and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.
  • the present invention comprises any one of miR-500a-3p or its expression promoting agent and an anticancer agent, and a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer agent resistant breast cancer to provide.
  • inhibiting the expression of LY6K or inhibiting miR-192-5p in breast cancer cells may restore sensitivity to anticancer drugs such as tamoxifen and may help smooth tumor treatment.
  • inducing expression of miR-500a-3p in breast cancer cells or adding miR-500a-3p may restore sensitivity to anticancer drugs such as tamoxifen, thereby helping to smooth tumor treatment.
  • LY6K expression is negatively correlated with ER ⁇ .
  • the relationship between ER ⁇ and LY6K mRNA, protein expression was tested by RT-PCR (A) and Western blotting (B).
  • h18S rRNA and ⁇ -actin are endogenously regulated (C and D).
  • Potential expression of LY6K in MCF7 and T47D lowers the transcriptional activity (C) and protein activity (D) of ER ⁇ .
  • Ago2 expression restores ER ⁇ expression.
  • LY6K gene expression levels were determined in mRNA and protein after 48 hours. mRNA and protein expression were analyzed by qRT-PCR (A) and Western blotting (B), respectively.
  • Figure 4 confirms the response to knockdown AGO2 following control and ER ⁇ treatment for 48 hours.
  • Hierarchical clustering data confirmed mature miRNAs based on miRNA microarray analysis of T47D / Mock cells compared to T47D / LY6K cells (A) and ESR1 3′UTR in miRanda (http://www.microrna.org/). MiRNA binding sites were predicted (B).
  • pri-miRNAs Primary miRNAs (pri-miRNAs) were upregulated in T47D / LY6K cells compared to T47D / Mock cells (A) and mature miRNAs induced by LY6K in ER ⁇ positive breast cancer cell lines (B).
  • Figure 7 identifies primary and mature miRNAs induced by ER ⁇ .
  • Hierarchical clustering data is based on miRNA microarray analysis between MCF7-ADR / Mock and MCF7-ADR / ER ⁇ .
  • Figure 8 identifies primary and mature miRNAs induced by ER ⁇ . Comparison of MCF7-ADR / Mock and MCF7-ADR / ER ⁇ by qRT-PCR confirmed up-regulation of primary-miRNAs (pri-miRA) expression (A) ER ⁇ negative breast cancer cell lines MCF7-ADR (left) and MDA-MB Mature miRNA induced by ER ⁇ at -468 (right) was identified (B).
  • ER ⁇ is a direct target of miR-192-5p.
  • ESR1 gene structure showing that the expected target site of miR-192-5p is at 3′UTR (A).
  • Luciferase assay in MCF7 cells showed miR-192-5p independent wild type (WT) ER ⁇ 3'UTR inhibition (B), while miR-192-5p mutant (MT) did not affect the inhibition of the expected binding site (C).
  • Luciferase assays in MCF7 cells showed that miR-29b-3p and miR-29c-5p showed little difference between the expected binding sites of wild type and mutant types (D and E).
  • LY6K is a direct target of miR-500a-3p and downregulates its expression.
  • the gene structure of LY6K shows that there are two predicted target sites of miR-500a-3p at 3'UTR (A).
  • Dual Luciferase assay in HEK293T cells shows LY6K (left) and miR-500a-3p (right) dependent inhibition of wild type (WT) LY6K 3'UTR, whereas mutant (MT) of seed sequence Luciferase activity is restored (B).
  • Ectopic expression of miR-500a-3p inhibits LY6K mRNA and protein expression in MCF7-ADR (left) and MDA-MB-468 (right) (C).
  • A represents miR-34a and miR-194-5p expected binding sites in the gene structure of LY6K 3′UTR, and miRNA binding sites of wild type (WT) and mutant (MT).
  • B in Figure 11 shows that miR-34a (left) and miR-194-5p (right) do not affect luciferase activity in HEK293T cells.
  • FIG. 12 shows regulation of ER ⁇ by miR-192-5p.
  • Transfection effect of miR-192-5p mimic in MCF7 and T47D cells (top). Potential expression of miR-192-5p downregulated ER ⁇ mRNA levels (bottom) (A). Re-expression of miR-192-5p in MCF7 and T47D cells inhibited the protein levels of ER ⁇ (B). MRNA and protein expression of LY6K in cell lines stabilized to overexpress human LY6K were measured by qRT-PCR and Western blot (C). Transfection efficiency of miR-192-5p inhibitor in T47D / LY6K cells stably expressing LY6K (left). miR-192-5p inhibition increased mRNA expression (right) (D). Reduced miR-192-5p expression inhibited ER ⁇ expression (E).
  • T47D / LY6K cells Three hours after treatment with varying amounts of tamoxifen, cell viability of T47D cells and T47D / LY6K cells was measured (A). In T47D / LY6K cells, transfection with a miR-negative control (NC) and a miR-192-5p inhibitor was performed 48 hours after transfection and cell viability was measured (B).
  • NC miR-negative control
  • Figure 14 shows that miR-500a-3p reexpression increases tamoxifen sensitivity in ER ⁇ negative breast cancer (A).
  • A Cell survival rate when treated with miR-negative control (NC) or miR-500a-3p after 24 hours incubation with different tamoxifen doses in MCF7-ADR. After 48 hours of treatment with carrier or tamoxifen caspase-3 activation was increased by miR-500a-3p (B).
  • ER in particular ER ⁇
  • ER ⁇ has an important effect in prescribing drugs suitable for breast cancer patients.
  • tumors were resistant to hormonal treatments such as tamoxifen and fulvestrant.
  • loss of ER ⁇ is one of the most important aspects of breast cancer.
  • ER ⁇ negatively correlates with the human LY6K gene.
  • ER ⁇ expression was downregulated when human LY6K gene was overexpressed in ER ⁇ positive cells. This reduction in ER ⁇ expression by LY6K can provide clues to the problem for hormone therapy resistant patients.
  • ER ⁇ promoter activity is inhibited and regulated by Twist, hMAPK. Regulation by miRNAs is also involved in epigenetic programs such as methylation and acetylation.
  • the miRNA microarrays of the invention showed that miR-192-5p is particularly overexpressed upon upregulation of human LY6K gene.
  • pri-miR-194-2 / 192 expression was upregulated in T47D cells transiently overexpressing LY6K.
  • the manner in which LY6K modulates the transcriptional activity of miR-192-5p is still unknown because the LY6K position is unclear.
  • these results indicate that the human LY6K gene can activate miR-192-5p at the transcription level and mature miR-192-5p inhibits ER ⁇ expression in ER ⁇ positive breast cancer cells.
  • miRNAs in breast cancer have been reported to be involved in ER ⁇ signaling and expression.
  • miR-192-5p has not been reported as breast cancer related miRNA in previous studies.
  • several studies have reported on miRNAs that act as ER ⁇ signaling inhibitors and target ER ⁇ directly, suggesting that these miRNAs may be involved in endocrine resistance in breast cancer.
  • ER ⁇ is a direct target of miR-192-5p. This suggests that miR-192-5p not only affects ER ⁇ signaling but also affects treatment resistance targeting ER ⁇ .
  • miR-192-5p As a potential target for esophageal cancer cells. Indeed, new cancer-related miRNAs (oncomiR), miR-519a, have been reported to confer tamoxifen resistance through cell proliferation and autologous cell death by targeting tumor suppressor genes. They have shown that induction of cell proliferation in breast cancer is associated with a decrease in autologous apoptosis with high doses of tamoxifen. We have observed that miR-192-5p induced by human LY6K gene in breast cancer cells is associated with tamoxifen resistance through cell proliferation.
  • oncomiR new cancer-related miRNAs
  • miR-519a have been reported to confer tamoxifen resistance through cell proliferation and autologous cell death by targeting tumor suppressor genes. They have shown that induction of cell proliferation in breast cancer is associated with a decrease in autologous apoptosis with high doses of tamoxifen.
  • the inhibition of miR-192-5p may be related to the apoptosis induced by tamoxifen, thus analyzing the more detailed relationship between miR-192-5p inhibition and apoptosis by tamoxifen regimen. There is a need.
  • ER ⁇ -induced miR-500a-3p in ER ⁇ -negative breast cancer cells directly regulates human LY6K expression and is effective in enhancing tamoxifen sensitivity.
  • miRNAs can play an important role as tumor genes or tumor suppressor genes, depending on the particular tumor subtype. In breast cancer, some miRNAs have been reported to affect tumorigenic processes and increase response to treatments such as anti-endocrine and chemotherapy.
  • miR-500a-3p was induced by ER ⁇ through miRNA microarray comparing MCF7-ADR / control with MCF7-ADR / ER ⁇ .
  • miR-500a-3p directly binds to the 3'UTR of human LY6K.
  • miR-500a-3p is involved in cell proliferation and metastasis and reduced LY6K expression in vitro.
  • LY6K is expressed at very high levels in ER ⁇ negative breast cancer.
  • ER ⁇ and LY6K have a negative correlation in breast cancer cells.
  • LY6K expression is downregulated by ectopic expression of ER ⁇ , which is an important target receptor for breast cancer target therapy.
  • Tamoxifen a selective estrogen antagonist
  • ER ⁇ positive breast cancer is well known as a medicament for ER ⁇ positive breast cancer and has no effect on ER ⁇ negative breast cancer patients.
  • Histone deacetylase (HDAC) inhibitors can re-express ER ⁇ transcriptional activity and improve endocrine therapy in combination with DNA damaging agents.
  • HDAC Histone deacetylase
  • CpG methylation of the estrogen receptor promoter by inhibiting DNA Methyltransferase (DNMT) activity can reactivate ER due to transcriptional silencing in ER ⁇ negative breast cancer cells.
  • ER reactivated by tamoxifen-binding recruits inhibitor complexes to regulate ER-reactive genes.
  • primary and mature miR-500a-3p is upregulated by ER ⁇ overexpression in ER ⁇ negative breast cancer cells.
  • miR-500a-3p was able to increase apoptosis observed by tamoxifen-induced cell viability and caspase-3 activation.
  • the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, including LY6K.
  • the present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting LY6K.
  • the present invention comprises the steps of analyzing the expression of LY6K in a sample isolated from the individual; And comparing the expression level with the LY6K expression level in the normal group.
  • the present invention also provides a pharmaceutical composition for enhancing the anticancer effect, comprising a LY6K inhibitor and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.
  • the LY6K inhibitor is one or more selected from the group consisting of a LY6K gene-specific siRNA, a LY6K gene-specific shRNA, a recombinant expression vector comprising a LY6K gene-specific siRNA, and a recombinant expression vector comprising a LY6K gene-specific shRNA. It is not limited.
  • the LY6K gene-specific siRNA is characterized in that represented by any one of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the LY6K gene specific shRNA is characterized in that represented by any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the expression vector is characterized in that any one selected from the group consisting of lentiviral vector, retrovirus vector and adenovirus vector.
  • the present invention includes a LY6K inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
  • the anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.
  • the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, comprising miR-192-5p.
  • the present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-192-5p.
  • the present invention comprises the steps of analyzing miR-192-5p expression in a sample isolated from the individual; And comparing the expression level with miR-192-5p expression level in the normal group, and provides an anticancer drug sensitivity diagnosis method for anticancer drug resistant breast cancer.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a miR-192-5p inhibitor and enhancing an anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.
  • the miR-192-5p inhibitor is any one of an anti-miR-192-5p oligonucleotide or an expression vector including the same, and the anti-miR-192-5p oligonucleotide is characterized in that represented by SEQ ID NO: 22.
  • the expression vector is any one selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors, but is not limited thereto.
  • the present invention includes a miR-192-5p inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating a cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
  • the anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.
  • the present invention also provides a biomarker composition for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer, comprising miR-500a-3p.
  • the present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-500a-3p.
  • the present invention comprises the steps of analyzing miR-500a-3p expression in a sample isolated from the individual; And comparing the expression level with miR-500a-3p expression levels in the normal group, and provides an anti-cancer drug sensitivity diagnostic method for anti-cancer drug-resistant breast cancer.
  • the present invention also provides a pharmaceutical composition for enhancing anticancer effect comprising miR-500a-3p or an expression promoter thereof and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.
  • the miR-500a-3p is characterized in that represented by SEQ ID NO: 23.
  • the expression promoter is any expression vector selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors expressing miR-500a-3p, but is not limited thereto.
  • the present invention includes any one of miR-500a-3p or its expression promoter and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
  • the anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.
  • RNA interference RNA interference
  • microRNA-mediated regulation RNA interference or microRNA technology
  • RNAs are produced by dicer enzymes belonging to the RNase III group, a dsRNA (double strandRNA) -specific endonuclease.
  • siRNA is a dsRNA consisting of about 21 nucleotides and consists of about 19 base pairs and two nucleotide overhangs present on the 3 'side. These 3 ′ overhangs function as intermediate mediators in the RNAi process. siRNAs are produced when long dsRNAs are made from transposons, viruses, or genes in vivo or by artificially injecting dsRNAs into cells from outside.
  • miRNAs are made naturally in cells, and are RNAs that do not have the genetic information to produce proteins.
  • the formation process of miRNA is as follows. It is transcribed into primary miRNAs by genes in the nucleus and then cleaved by drosha to form precursor miRNAs in the form of short hairpins, which then migrate from the nucleus to the cytoplasm. In the cytoplasm, a small stem loop of a precursor miRNA is cleaved by a diser enzyme, a kind of ribonuclease, and matures into a single strand of miRNA.
  • siRNA inhibits gene function by inducing degradation of RNA through the RNA-induced silencing complex (RISC), while miRNA inhibits the translation of mRNA, which is part of the nucleotide sequence of the 3'-UTR (untranslated region) of the target mRNA.
  • RISC RNA-induced silencing complex
  • Complementary binding inhibits gene function by inhibiting the translation of mRNA into proteins in ribosomes.
  • RNAi using synthetic siRNA is a specific nucleic acid sequence in a short time by transfection of dsRNA to a target cell directly to the cell in vitro. There is an advantage that can be screened for the effect of inhibiting gene expression.
  • RNAi using short hairpin RNA instead of directly injecting synthetic dsRNA, plasmid DNA is used to transcribe single-stranded RNA of hairpin structure, and shRNA transcribed from the DNA injected into the cell undergoes a process similar to that of microRNA. Finally converted to siRNA. Specifically, in the case of RNAi using shRNA, the plasmid controlled by the RNA polymerase III promoter is transcribed into single-stranded RNA having a loop similar to pre-miRNA in the nucleus of the target cell, and then moved to the cytoplasm by Exportin-5. It is converted into siRNA during processing by Dicer.
  • shRNA short hairpin RNA
  • RNA capable of artificially expressing the shRNA is recombinantly injected into the cell. Thereafter, the shRNA is expressed in the cell by the expression mechanism of the miRNA present in the cell, thereby suppressing the expression of the target gene.
  • RNA is sometimes called "artificial microRNA”.
  • the miRNA expression vector can be transformed or infected into a cell so that the miRNA of the present invention can be expressed temporarily or permanently in the cell.
  • the recombinant expression vector of the present invention may be constructed by recombinant DNA methods known in the art.
  • the expression vector may be selected from the group consisting of plasmids, lentiviral vectors, retroviral vectors, adenovirus vectors known in the art, used for replication and expression of mammalian cells or other target cell types.
  • viral vectors in the present invention are particularly preferred. The reason for this is that viral vectors have high efficiency of nucleic acid delivery in vivo, and therefore, the effect is expected to be high when applied to RNAi.
  • Lentiviruses are a type of retroviral vector that can transduce even non-dividing cells, and the injected gene can be expressed for several months or more, which is advantageous in maintaining RNAi for a long time.
  • the present invention also provides a transformed cell prepared by introducing the expression vector into a host cell.
  • the expression vector into which the gene encoding the miRNA sequence is inserted can be introduced into a host cell by methods well known to those skilled in the art. Introduction methods include, but are not limited to, electroporation and lipofection, and methods known in the art may be selected.
  • the shRNA or siRNA can be designed by putting the sequence of the target gene in the Internet site such as Ambion, invitrogen, RNAi codex.
  • the Gregory Hannon lab site also designs siRNAs or shRNAs for these genes, and targets genes in other sites and programs, including http://www.dharmacon.com/designcenter/designcenterpage.aspx and Invivogen's siRNA Wizard v3.1. Inserting a sequence allows the design of shRNA or siRNA sequences.
  • the shRNA or siRNA sequence for the target gene is not limited and can be easily designed by those skilled in the art through various sites and programs as described above.
  • Exemplary LY6K siRNA sequences of the present invention include 5'-GCAAAUGGACAGAGCCAUA-3 ', 5'-ACAAUAGAGUGUGGUGUCAUGUUUG-3', 5'-CAAAUGGACAGAGCCAUACUGCGUU-3 'and the like.
  • the LY6K siRNA sequence of the present invention is not limited to the three sequences exemplified above.
  • the LY6K shRNA sequences of the present invention include 5'-UGUGGACAGACGCCAACCUGACUGCGAGA-3 ', 5'-GCAAAUGGACAGAGCCAUACUGCGUUAUA-3', 5'-GAGGAGAAGCGGUUUCUCCUGGAAGAGGCC-3 ', and 5'-UCCUGCUGCUGGCCUC shUCC-3, etc. It is not limited to the sequences exemplified above.
  • miR-192-5p anti-oligonucleotide sequence of the present invention is the same as 5'-GGC TGT CAA TTC ATA GGT CAG-3 '.
  • miR-500a-3p sequence of the present invention is the same as 5'-AUGCACCUGGGCAAGGAUUCUG-3 '.
  • the composition according to the present invention preferably comprises a gene carrier or cell comprising LY6K siRNA, LY6K shRNA or miR-500a-3p, but is not limited thereto.
  • the gene carrier is preferably a vector or a recombinant virus, but is not limited thereto.
  • the vector may be a linear DNA vector, a plasmid vector, a vector containing a viral expression vector or a recombinant retrovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus expressed in human or animal cells.
  • a recombinant viral vector including a virus, AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector.
  • the recombinant virus is preferably a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus or lenti virus, but is not limited thereto.
  • the composition may be administered parenterally during clinical administration, and may be administered by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.
  • the composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
  • the daily dosage of the composition is about 0.0001 to 1000 mg / kg, specifically 0.001 to 100 mg / kg, can be administered once or several times a day, but the weight, age, sex, health, diet, administration of the patient The range varies depending on the time, the method of administration, the rate of excretion and the severity of the disease.
  • Vectors containing LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention specifically contain 0.01 to 500 mg, more specifically 0.1 to 300 mg, LY6K siRNA, LY6K shRNA or miR- for recombinant viruses containing 500a-3p, specifically 10 3 ⁇ 10 12 IU contain (10 to 10 10 PFU), one or more specifically, contains 10 5 to 10 10 IU, but not limited thereto.
  • the cell containing the LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention specifically contains 10 3 to 10 8 , more specifically contains 10 4 to 10 7 , It is not limited.
  • the effective dose of the composition containing a vector or cells comprising LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention as an active ingredient is 0.05 to 12.5 mg / kg for a vector per kg of body weight, recombinant virus In the case of 10 7 to 10 11 virus particles (10 5 to 10 9 IU) / kg, 10 3 to 10 6 cells / kg for the cells, specifically 0.1 to 10 mg / kg for the vector, recombinant virus In the case of 10 8 to 10 10 particles (10 6 to 10 8 IU) / kg, in the case of cells 10 2 to 10 5 cells / kg, it can be administered 2-3 times a day.
  • the composition is not necessarily limited thereto, and may vary depending on the condition of the patient and the degree of onset.
  • compositions can be administered in various parenteral formulations during actual clinical administration, when formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like commonly used.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • MCF7, MCF7 / ADR, MDA-MB-468 and T47D cells were cultured in DMEM (WelGENE, Korea) medium containing 10% fetal calf serum (FBS, Gibco). These cells were maintained at 37 ° C in humid conditions in 5% CO 2 and 95% air. T47D cell line stably overexpressing the human LY6K gene was made with G418 selection.
  • MCF7 and T47D cells were transfected for 48 h with hLY6K / pCMV6-infiltrating clones purchased from Origene using FuGENE® preparation (Promega).
  • MCF7-ADR and MDA-MB-468 were transfected with human ER ⁇ pCMV plasmid using Fugene® reagent (Promega) according to the manufacturer's instructions.
  • MCF7 and T47D cells were transfected with miR-192-5p mimics (miRVana TM miRNA mimic, ambion) for 48 hours using siPORT TM NeoFX TM transfection agent (Ambion).
  • Controls were transfected with negative control miRNA mimics (miVana TM miRNA mimic, Negative control # 1, ambion).
  • MCF7 and T47D cells were transfected with 10 nM Argonatue 2 siRNA (SMARTpooled, Dharmacon) and 4 ⁇ g of hLY6K / pCMV6 clone using Lipofectamin 2000 (Invitrogen TM) according to the manufacturer's instructions.
  • MCF7-ADR cells were transfected with 10 nM Argonatue 2 siRNA (Dharmacon, USA) and 5 ⁇ g of human ER ⁇ clones using Lipofectamin 2000 (Invitrogen, USA) for 48 hours as directed by the manufacturer. Transfection with scrambled siRNA (Dharmacon, USA) and pCMV-Flag plasmid is a control experiment. Subsequent experiments were performed 48 hours after transfection. After 48 hours, each was separated and used for qRT-PCR and Western blotting.
  • qRT-PCR Quantitative RT-PCR
  • Proteins were extracted using NucleoSpin® RNA / Protein kit (MACHEREY-NAGEL). Proteins were analyzed using BCA (Bicinchoninic acid) solution (Sigma, B9643) and copper sulfate solution (Sigma, C2284). Proteins were SDS-PAGE (12% separation gel, 5% accumulation gel; H 2 O, 30% acrylamide (Bio-Rad, # 161-0156), 1.5 M pH 8.8 Tris, 10% SDS, 10% and Ammonium persulfate, TEMED (Sigma, T9281) was separated and transferred to a clean membrane (Atto, AE-6667-P) The antibodies used in the experiments are shown in Table 2. The immunoreactive proteins were horseshoe. Dish peroxidase was detected with a bound secondary antibody and amplified with a chemiluminescent material called EzWestLumi plus (ATTO, JAPAN) ⁇ -actin was used as loading control.
  • 3'UTR of human ER ⁇ was amplified by PCR from T47D cDNA and cloned into the XbaI site of pGL3-control vector (Promega). Seed sequences miR-29b, miR-29c and miR-192 on the 3'UTR of human ER ⁇ were mutated with a PCR based approach.
  • T47D and MCF7 cells were simultaneously transfected with Lipofectamin 2000 (Invitrogen) with luciferase constructs (1.8 ⁇ g reporter gene / well in 6 well plates) and pCMV-LY6K vector (200 ng / well in 6 well plates). .
  • Human LY6K 3'UTR containing the expected miR-500a-3p seed sequence was amplified by PCR from human genomic DNA and psiCHECK TM -2 vector (Promega, USA) using an In-fusion® HD cloning kit (Clontech Laboratories, USA) ) The miR-500a-3p seed sequence on LY6K 3'UTR was mutated using the QuickChange II XL site-directed mutation kit (Agilent Technologies, USA).
  • HEK293T cells were transfected using a Lipofectamin 2000 (Invitrogen, USA) with a luciferase reporter construct containing LY6K 3'UTR variant and 30 nM miR-500a-3p mimics (mimics) or negative control miRNAs. . After 48 hours, cells were manually lysed and measured with a dual luciferase assay system (Promega, USA).
  • MCF7-ADR, MDA-MB-468 cells, T47D or T47D / LY6K cells were seeded in 1 ⁇ 10 4 cells / well in 96 well plates.
  • Cells were treated with tamoxifen (Sigma, H790) after incubation for 24 hours and incubated again for three hours.
  • Cells were seeded and transfected with siPORTTM NeoFXTM transfection preparation (Ambion) with negative miRNA mimics or mature miR-192-5p inhibitors (30 nM, ambion).
  • tamoxifen (4-Hydroxytamoxifen; "4-OHT”; Sigma, USA) at a concentration of 1-10 ⁇ M.
  • WST-8 (Enzo0R) labeling mixtures were added to each well and quantified using a scanning multi-well spectrophotometer according to the manufacturer's instructions.
  • MCF7-ADR cells were seeded in 100 mm dishes for 24 hours using siPORT TM neoTX TM transfection Agent (Ambion, USA) with miR-500a-3p mimics (mimics) or negative control miRNAs. Then, 4-hydroxytamoxifen (4-OHT) was treated to a final concentration of 30 nM. After 48 hours, the cells were lysed and caspase-3 activity was measured on the cell lysate according to the manufacturer's instructions with the Caspase-3 / CPP32 Colorimetric Assay Kit (Biovision, USA).
  • LY6K siRNA sequence of the configuration of the present invention is not limited only to the following sequences, and may be generated using a sequence generation program as necessary.
  • LY6K siRNA Name Primer Number LY6K siRNA (1) 5'-GCAAAUGGACAGAGCCAUA-3 ' 15 LY6K siRNA (2) 5'-ACAAUAGAGUGUGGUGUCAUGUUUG-3 ' 16 LY6K siRNA (3) 5'-CAAAUGGACAGAGCCAUACUGCGUU-3 ' 17 LY6K shRNA (1) 5'-UGUGGACAGACGCCAACCUGACUGCGAGA-3 ' 18 LY6K shRNA (2) 5'-GCAAAUGGACAGAGCCAUACUGCGUUAUA-3 ' 19 LY6K shRNA (3) 5'-GAGGAGAAGCGGUUUCUCCUGGAAGAGCC-3 ' 20 LY6K shRNA (4) 5'-UCCUGCUGCUGGCCUCCAUUGCAGCCGGC-3 ' 21 anti-miR-192-5pnucleotide 5'- GGC TGT CAA TTC ATA GGT CAG -3 ' 22 miR-500a-3p 5'-AUGCACC
  • LY6K expression is negatively correlated with ER ⁇ .
  • ER ⁇ mRNA and protein were downregulated by LY6K (FIG. 1C, D).
  • the expression of LY6K was decreased by the ectopic expression of ER ⁇ in MCF7-ADR and MDA-MB-468, which are well known as ER ⁇ negative breast cancer cells.
  • Ectopic expression of ER ⁇ decreased both LY6K mRNA and protein expression in ER ⁇ negative breast cancer cells (FIGS. 2A, B).
  • LY6K regulates ER ⁇ expression after transcription.
  • LY6K affects miRNAs expression in ER ⁇ positive cells.
  • miRNA microarray analysis was performed on T47D, an ER ⁇ positive cell as compared to T47D / LY6Ks. Unsupervised hierarchical clustering showed a clear expression pattern between T47D / Mock and T47D / LY6K (FIG. 5A).
  • miRNAs upregulated in T47D / LY6K using miRanda hsa-miR-29-3p, hsa-miR-29c-5p and hsa-miR-192-5p bind to the 3'UTR of the human ESR1 gene ( 5B).
  • miRNA genes are transcribed by RNA polymerase II and primary miRNAs (pri-miRNAs) are activated by binding of polymerase to the DNA sequence of interest (Lee, Kim et al. 2004).
  • pri-miRNAs primary miRNAs
  • LY6K primary miRNAs
  • miRNA microarray analysis was performed by comparing MCF7-ADR, an ER ⁇ negative cell, with MCF7-ADR / ER ⁇ , which transiently overexpresses ER ⁇ .
  • MCF7-ADR MCF7-ADR / ER ⁇
  • ER ⁇ ER ⁇
  • FIG. 7 shows that several miRNAs were found to be affected and upregulated by ER ⁇ .
  • miR-192-5p directly targets the ESR1 gene.
  • the luciferase reporter construct ESR1 3'UTR containing each miRNA predicted binding site was pGL3-controlled.
  • the vector was cloned (FIG. 9A).
  • Luciferase activity in MCF7 cells was reduced by transpotent expression of LY6K, transfected luciferase reporter construct wild type or miR-192-5p mutant type (FIG. 9B).
  • ESR1 is a direct target of miR-192-5p
  • the miR-192-5p mimic and the wild type luciferase reporter construct or mutant were transfected together in MCF7 cells.
  • ESR1 mutations at the expected miR-192-5p target sites did not affect relative luciferase activity (FIG. 9C). In contrast, luciferase activity was restored in the mutant ESR1 luciferase reporter construct.
  • phosphorus In beats ER ⁇ Induced by miR -500a-3p is human LY6K Regulate by targeting genes.
  • the 3'UTR portion of the LY6K gene was examined to find miRNA binding motifs using the miRnada program to determine if the decrease in LY6K expression was due to direct targeting among three selected miRNAs.
  • Luciferase reporter constructs were prepared in which human LY6K 3′UTR containing each miRNA predicted binding site was inserted into a psiCHECK-2 vector (FIGS. 10A and 11A). Only one miR-500a-3p overexpression of these miRNAs reduced luciferase activity (FIG. 10B, right). Luciferase activity was also decreased by ectopic expression of ER ⁇ (FIG. 10B, left).
  • ER ⁇ positive cell lines MCF7 and T47D were transiently transfected for 48 hours with miR-192-5p mimic and negative control mock (NC).
  • N miR-192-5p mimic and negative control mock
  • qRT-PCR confirmed the transfection efficiency of miR-192-5p mimics in MCF7 and T47D cells (FIG. 12A, top).
  • ER ⁇ mRNA was significantly reduced by overexpression of miR-192-5p (FIG. 12A, bottom).
  • results similar to ER ⁇ protein expression for overexpression of miR-192-5p mimics in MCF7 and T47D cells were confirmed (FIG. 12B).
  • miR-192-5p downregulated ER ⁇ expression by directly targeting the 3′UTR. From this, it can be hypothesized that miR-192-5p inhibition can restore tamoxifen resistance in cells overexpressing LY6K. This is because tamoxifen is well known as a drug targeting ER ⁇ .
  • T47D / LY6K cells exhibited tamoxifen resistance
  • T47D and T47D / LY6K cells were treated with tamoxifen by concentration and cell viability was observed. As a result, tamoxifen treatment significantly reduced cell viability in T47D cells, which are ER ⁇ positive breast cancer cells.
  • T47D / LY6K cells when treated with tamoxifen, cell survival was hardly reduced due to downregulation of ER ⁇ expression (FIG. 13A).
  • inhibition of miR-192-5p expression compared to negative miRNA inhibitors (NC) resulted in T47D / LY6K cells becoming more sensitive to tamoxifen (FIG. 13B). From this result, it can be seen that miR-192-5p regulates tamoxifen resistance through cell proliferation.
  • miR-500a-3p lowered LY6K expression by directly targeting the 3′UTR of LY6K.
  • miR-500a-3p enhances tamoxifen sensitivity through apoptosis activation in ER ⁇ negative breast cancer cells.
  • Tamoxifen is a selective estrogen antagonist and is well known as a drug targeting ER ⁇ in ER ⁇ positive breast cancer patients, but only about 10-15% of patients with ER ⁇ negative breast cancer respond to tamoxifen.
  • the present inventors treated 4-OHT (4-hydroxytamoxifen) by concentration in MCF7-ADR cells transiently overexpressing ER ⁇ and observed cell viability.
  • Cell viability in MCF7-ADR was significantly reduced by ectopic expression of miR-500a-3p (FIG. 14).
  • tamoxifen in breast cancer cells not only inhibited cell proliferation but also induced apoptosis.
  • miR-500a-3p reexpression enhances tamoxifen sensitivity through apoptosis.
  • miR-500a-3p significantly increased caspase-3 activation when tamoxifen treated as compared to the negative control.
  • miR-500a-3p induced by ER ⁇ regulates tamoxifen sensitivity by targeting LY6K in ER ⁇ negative breast cancer cells.

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Abstract

La présente invention concerne une composition de biomarqueur pour le diagnostic de la sensibilité à un agent anticancéreux dans le cancer du sein résistant à un agent anticancéreux, un kit de diagnostic, un procédé de diagnostic ou une composition pharmaceutique pour favoriser des effets anticancéreux, permettant de soutenir un traitement efficace de tumeurs par le rétablissement de la sensibilité à des agents anticancéreux, tels que le tamoxifène, etc. si l'expression de LY6K est inhibée ou si miR-192-5p est supprimé dans les cellules du cancer du sein. De même, il est possible de soutenir un traitement efficace de tumeurs par le rétablissement de la sensibilité à des agents anticancéreux tels que le tamoxifène, etc. si l'expression de miR-500a-3p est induite ou si miR-500a-3p est administré dans les cellules du cancer du sein.
PCT/KR2016/002215 2015-03-04 2016-03-04 Composition de biomarqueur pour le diagnostic de la sensibilité à un agent anticancéreux dans le cancer du sein résistant à un agent anticancéreux WO2016140552A1 (fr)

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KR10-2015-0030254 2015-03-04
KR1020150030254A KR101667649B1 (ko) 2015-03-04 2015-03-04 LY6K 발현 저해제 또는 miR-192-5p 저해제를 포함하는 항암제 내성 유방암 치료 보조제
KR1020150113745A KR101581721B1 (ko) 2015-08-12 2015-08-12 miR-500a-3p를 포함하는 항암제 내성 유방암 치료 보조제 및 miR-500a-3p를 발현하거나 가하여 항암제 내성 유방암 환자의 항암제 민감성을 회복하는 방법
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CN110787181A (zh) * 2019-12-10 2020-02-14 合肥市第二人民医院 一组miRNA及其在生物靶向治疗乳腺癌中的应用
EP3707127A4 (fr) * 2017-11-09 2021-12-01 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Composés pour inhiber ly6k et leurs procédés d'utilisation

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WO2014145142A2 (fr) * 2013-03-15 2014-09-18 Miles Gregory Procédé d'amélioration de la survie dans le cancer

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3707127A4 (fr) * 2017-11-09 2021-12-01 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Composés pour inhiber ly6k et leurs procédés d'utilisation
CN110787181A (zh) * 2019-12-10 2020-02-14 合肥市第二人民医院 一组miRNA及其在生物靶向治疗乳腺癌中的应用

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