KR20190112977A - Use of HSP90AA1 for Treating or Diagnosing NANOG-mediated Diseases - Google Patents

Abstract

The present invention relates to the use of HSP90AA1 for treating or diagnosing NANOG-mediated diseases. More specifically, the present invention relates to the use of HSP90AA1 for treating and diagnosing anti-cancer immune resistant cancer with increased NANOG expression. A composition for treating NANOG dependent resistant cancer using a novel NANOG/HSP90AA1 immunoresistant molecular axis as a pathological marker, the resistant cancer diagnosing kit or an information providing method for diagnosis according to the present invention is very useful for more precisely and quickly diagnosing and treating cancer that is resistant to chemotherapy, such as chemotherapy or immunotherapy and cancer that is relapsed or metastasized after chemotherapy.

Description

Use of HSP90AA1 for Treating or Diagnosing NANOG-Mediated Diseases

The present invention relates to the use of HSP90AA1 (heat shock protein AA1) for the treatment or diagnosis of NANOG-mediated diseases, and more particularly to the use of HSP90AA1 for the treatment and diagnosis of cancer-resistant cancers with increased NANOG expression will be.

Surgical surgery, radiation therapy, anticancer therapy, and immunotherapy for the treatment of cancer include, but despite the many treatments, the clinical efficacy is low because of recurrence of cancer. The main cause of recurrence, a clinical challenge, is the growth of cancer cells with more malignant characteristics due to the inherent resistance of the cancer as well as the treatment resistance due to acquisition tolerance that occurs after various chemotherapy. Therefore, for effective cancer treatment, research on overcoming resistance obtained by various chemotherapy as well as inherent resistance of cancer is essential. In the study of overcoming resistance, cancer treatments should be studied to identify resistance-related genes or molecular axes and diagnose and target using identified genes or molecular axes.

HSP90 (heat shock protein 90) has been shown to induce degradation of tumor-induced proteins through function inhibition, which has proven its function as a target anticancer agent. HSP90 inhibitors have entered clinical trials since 1999, but frequent resistance is a problem. Therefore, attention should be paid to the detailed and specific function of HSP90.

Among several subtypes of HSP90, HSP90AA1 has been reported to increase expression when exposed to stress and to be highly expressed in leukemia, breast cancer and pancreatic cancer ( Yufu et al., Leuk Res. 16 (6-7): 597-605). , 1992; Tian et al., Gene. 542 (2): 122-8, 2014, Jameel et al., Int J Cancer. 50 (3): 409-15, 1992; Gress et al., Cancer Res. 54 (2): 547-51, 1994 ) Therefore, stress-sensitive HSP90AA1 may play an important role in early tumor cells produced by immune evasion against stress caused by early oncogenic stimuli during cancer development. In fact, it has been reported that this HSP90AA1 may promote the resistance of the anticancer drug cisplatin due to overexpression in ovarian cancer cells ( Chu et al., Mol Biol Rep. 40 (1): 1-6, 2013 ), HSP90AA1 is known to promote cancer metastasis by binding to MMP2 to stabilize the protein ( Eustace et al., Nat Cell Biol . 6 (6): 507-14, 2004; Wang et al., Proc Natl Acad Sci US A. 106 (50): 21288-93, 2009 ).

In a previous study, we identified the molecular axis related to the anticancer immunity resistance of NANOG / NATG / TCL1 / AKT / MCL-1, in which NANOG is an essential molecule for obtaining immunity (Korea Patent Publication 10-2012-0083504).

Accordingly, the present inventors have identified a molecular axis that may be a direct target of recurrent cancer, resistant cancer, or metastatic cancer after anticancer immunity treatment, and thus, in order to effectively diagnose and treat NANOG dependent resistant cancer, Among the several subtypes of HSP90, the expression of HSP90AA1 was unspecifically increased, and the targeting and availability of HSP90AA1 in the treatment of NANOG mediated anticancer immunity was investigated. As a result, it was confirmed that the expression of HSP90AA1 is directly regulated by the stem cell factor NANOG, confirming that the increased HSP90AA1 is an essential molecule for the acquisition of immunity in tumor cells obtained anticancer immunity, and completed the present invention. .

An object of the present invention is to target HSP90AA1 of the NANOG / HSP90AA1 immunoresistant molecule axis, and to prevent or treat NANOG dependent resistant cancer for treating or diagnosing cancer-resistant cancer with increased NANOG expression. To provide a way to provide information.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of NANOG dependent resistant cancer containing HSP90AA1 expression or activity inhibitor as an active ingredient.

The present invention also provides a kit for diagnosing NANOG dependent resistant cancer, comprising a probe capable of amplifying the HSP90AA1 gene or a probe capable of hybridizing with the HSP90AA1 gene under strict conditions.

The present invention also provides a method of measuring the mRNA level or HSP90AA1 protein level of HSP90AA1 gene from an isolated biological sample; And it provides a method for providing information for the diagnosis of NANOG dependent resistant cancer comprising comparing the measured mRNA level or HSP90AA1 protein level of the HSP90AA1 gene with a control sample.

The composition for the treatment of NANOG dependent resistant cancer using the novel NANOG / HSP90AA1 immunoresistant molecular axis as a pathological marker according to the present invention, the resistant cancer diagnostic kit or a method for providing information for diagnosis is resistant to chemotherapy such as an anticancer agent or immunotherapy. It is very useful for more precisely and quickly diagnosing and treating cancer in which recurrence or metastasis has occurred after this cancer and chemotherapy.

1 shows the expression (A) and the correlation (B) of HSP90AA1 protein and NANOG protein in various human cancer cell lines.
Figure 2 shows the correlation between the expression of HSP90AA1 protein and NANOG according to the progression of human cervical cancer, and treatment resistance and survival rate according to the expression of the two proteins.
Figure 3 confirms the expression of nonspecifically increased HSP90AA1 in anticancer immune resistant CaSki P3 cell lines, and plays a role of HSP90AA1 in cisplatin anticancer drug resistance, radiation resistance and tumorigenic capacity, including anticancer immune resistance of anticancer immune resistant CaSki P3 cells. It is confirmed.
4 shows that HSP90AA1 expression is decreased when NANOG expression is inhibited in the anti-cancer immune resistant CaSki P3 cell line, and that HSP90AA1 expression is increased when NANOG is overexpressed in the primary cancer cell line CaSki, and that HSP90AA1 expression is decreased when NANOG is overexpressed in cervical cancer cells. Induction of apoptosis factor MCL-1 and expression of the cell proliferation factor CyclinA was induced to confirm cisplatin anticancer drug resistance, radiation resistance and tumor cell formation ability including anticancer immunity resistance.
Figure 5 shows the expression of HSP90AA1 protein dependent on the transcriptional activity of NANOG in HEK293 cell line, the expression of HSP90AA1 mRNA, the identification of NANOG binding site in the HSP90AA1 promoter, the increase of HSP90AA1 promoter activity dependent on the transcriptional activity of NANOG, and the NANOG protein in the HSP90AA1 promoter. It is confirmed that this is directly coupled.
Figure 6 confirms that NANOG of the HSP90AA1 inhibitor AUY-922 modulates immunity, chemistry, radiation resistance and cancer stem cell-like properties through the ANOG / HSP90AA1 / AKT pathway in various human cancer cell lines overexpressed.
Figure 7 confirms that the inhibitory effect is increased by the inhibitor AUY-922 of HSP90AA1 in the immunotherapy model for human cancer cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present specification, "primary cancer" means a conventional cancer, "resistant cancer" means an anticancer drug or cancer resistant to cancer, and "anticancer immunity cancer" refers to a cancer obtained resistance to chemotherapy. In addition, anticancer immunotherapy refers to the integration of all systems of inducing immune responses against cancer specific antigens or cancer related antigens to remove cancer cells by cancer specific toxic immune cells. Immune induction methods for cancer antigens include genes, proteins, viruses, dendritic cells and the like. "Recurrence cancer" refers to cancer that has reoccurred after conventional cancer treatment, and "resistant cancer" refers to cancer that is resistant to the cancer treatment. Here, conventional cancer treatments include, for example, surgery, chemotherapy, radiation therapy, hormonal therapy, biological therapy, immunotherapy and the like. Examples of the cancer include brain tumors, spinal cord tumors, retinal cell tumors, oral cancers, nasal cancers, sinus cancers, pharyngeal cancers, laryngeal cancers, head and neck cancers including cervical cancers, skin cancers, breast cancers, thyroid cancers, breast cancers including malignant adrenal tumors Gastrointestinal cancer, kidney cancer, bladder cancer, prostate cancer, including lung cancer, pleural tumor, respiratory cancer, esophageal cancer, stomach cancer, small intestine malignant tumor, colon cancer, anal cancer, liver cancer, biliary tract cancer or pancreatic cancer Urinary cancer including testicular cancer or penile cancer, cervical cancer, cervical cancer, gynecological cancer including choriocarcinoma or ovarian cancer, hematologic cancer including acute or chronic leukemia, malignant lymphoma or multiple myeloma, bone tumor or soft tumor Bone or soft tumors, and pediatric cancer including pediatric leukemia or pediatric solid tumors. In addition, the term "cancer metastasis or metastatic cancer" refers to the metastasis of a primary or recurring cancer occurring at a specific site to another site.

It is known that NANOG is an essential molecule for obtaining anticancer immunity (Korea Patent Publication No. 10-2012-0083504). It was confirmed in the present invention that the expression of HSP90AA1 is non-specifically increased in this immunotolerant mechanism by NANOG.

Since the HSP90AA1 protein of the present invention shows not only anticancer immunity but also anticancer agent and radiation resistance, and is highly expressed in high metastatic cancer cells, NANOG-mediated NANOG-dependent by administering an inhibitor that inhibits the expression or activity of the gene or protein of the protein Recurrent cancer, resistant cancer or cancer metastasis can be used for therapeutic purposes as well as recurrence and metastasis of anticancer immune resistant cancer.

The prevention or treatment of the recurrence and metastasis of anticancer immune cancer of the present invention includes the prevention, inhibition or alleviation of the recurrence and metastasis of anticancer immune cancer.

Therefore, in one aspect, the present invention relates to a composition for preventing or treating NANOG dependent resistant cancer, which contains an inhibitor of HSP90AA1 expression or activity as an active ingredient.

Examples of the HSP90AA1 gene inhibitor (expression inhibitor) of the present invention include siRNA, antisense, or shRNAi that specifically bind to mRNA of the HSP90AA1 gene and inhibit expression. Since the HSP90AA1 gene exhibits anticancer immunity resistance, anticancer agent and radiation resistance, and is related to cancer metastasis, the HSP90AA1 gene may prevent or treat relapse and metastasis of NANOG dependent resistant cancer by inhibiting NANOG gene expression by siRNA, antisense, or shRNAi. have.

As used herein, "siRNA" refers to a double-chain RNA that induces RNA interference through cleavage of mRNA of a target gene, and an RNA strand of a sense sequence having the same sequence as the mRNA of the target gene and an antisense having a complementary sequence thereto. It consists of the RNA strand of the sequence.

The siRNA may include a form expressed by inserting the siRNA itself or a base sequence encoding the siRNA synthesized in vitro into the expression vector.

The siRNA may preferably have one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 5.

More specifically, siRNA sequences set forth in SEQ ID NOS: 1 to 5 are shown in Table 1 below.

In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the HSP90AA1 gene or fragment thereof, and can bind to the mRNA to inhibit the expression of the cancer metastasis gene or fragment.

The inhibitor of the HSP90AA1 protein may be a substance that inhibits the expression or function of the protein, specifically, may be a monoclonal antibody or polyclonal antibody of the HSP90AA1 protein, most preferably AUY -922, but is not limited to such.

The AUY-922 is known to target HSP90 alpha at low concentrations (IC50 concentration 13 nM).

In addition, the composition for the prevention or treatment of recurrence and metastasis of the anticancer immunity cancer of the present invention containing the mRNA or protein inhibitor of the HSP90AA1 gene as an active ingredient further comprises one or more anticancer agents or immunotherapy agents, or Can be used.

Combination with the composition of the present invention may be characterized by simultaneous, separate or sequential administration.

Inhibitors or antibodies of the HSP90AA1 protein can be used in conjunction with chemotherapeutic agents or radiation therapy well known to those skilled in the art, for example cyclophosphamide, aziridine, alkylalconsulfonates, nitrosoureas, Alkylating agents such as dacarbazine, carboplatin, cisplatin and the like, antibiotics such as mitomycin C, anthracycline, doxorubicin (adriamycin), and antimetabolitic such as methotrexate, 5-fluorouracil, cytarabine, etc. agent), plant-derived drugs such as vinca alkaloids, and hormones.

In addition, the composition for the prevention or treatment of the NANOG-dependent resistant cancer of the present invention containing the mRNA or protein inhibitor of the HSP90AA1 gene as an active ingredient, in addition to the active ingredient described above for administration to one other pharmaceutically acceptable carrier. It can be preferably formulated into a pharmaceutical composition including the above.

Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.

In addition, the composition for the prevention or treatment of NANOG dependent resistant cancer of the present invention containing the mRNA or protein inhibitor of the HSP90AA1 gene as an active ingredient may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient. The auxiliary agent may be a solubilizer such as an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, a lubricant, or a flavoring agent.

In addition, the composition for the prevention or treatment of NANOG dependent resistant cancer of the present invention may be added in addition to diluents, dispersants, surfactants, binders and lubricants to injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or It may be formulated into a tablet. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

In addition, the composition for the prevention or treatment of cancer-resistant cancer of the present invention is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, Administration can be in conventional manner via the intraocular or intradermal route.

The dosage of the composition for preventing or treating cancer-resistant cancer is meant an amount required to achieve an effect of suppressing the recurrence and metastasis of the cancer-resistant cancer. Thus, the type of disease, the severity of the disease, the type and content of the active ingredients and other ingredients contained in the composition, the type of formulation and the age, body weight, general health status, sex and diet of the patient, time of administration, route of administration and composition It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. In adults, when the inhibitor of the HSP90AA1 gene is administered once or several times a day, 0.01 ng / kg to 10 mg / kg for siRNA, 0.1 ng / kg to 10 mg / kg for compound, and monoclonal antibody It is preferable to administer at a dose of 0.1ng / kg-10mg / kg.

In another aspect, the present invention relates to a kit for diagnosing NANOG dependent resistant cancer comprising a probe capable of amplifying HSP90AA1 gene or a probe capable of hybridizing with HSP90AA1 gene.

As used herein, the term “probe” is a concept that encompasses antibodies, PCR primers, nucleic acid probes, and the like, which specifically bind to and recognize HSP90AA1 gene or protein.

In the present invention, the primer for the HSP90AA1 gene is preferably SEQ ID NO: 6 or SEQ ID NO: 7, but is not limited thereto.

The diagnostic kit for NANOG dependent resistant cancer of the present invention is for detecting a recurrence and metastatic pathological marker of anticancer immune cancer in a physiological sample, wherein the HSP90AA1 gene is specific to inside and outside cancer cells having recurrence and metastasis of anticancer immune cancer. As the expression rate increases, and the tumorigenic ability of the cell increases in proportion to its expression level, the recurrence of anti-cancer immunity cancer is quantified by quantitatively analyzing the expression level of the HSP90AA1 gene or the protein encoded therein in cancer cells or tissues. And metastasis.

The HSP90AA1 is derived from human or mouse, and known sequences, such as GenBank Accession No. NM_005348 (Person), GenBank Accession No. NM_010480 (mouse) or the like, or may include a variant capable of performing the same function even if deleted, substituted, added to the base or amino acid.

The composition for diagnosing recurrence and metastasis of anticancer immune cancer of the present invention may include a monoclonal antibody or a polyclonal antibody that specifically binds to an epitope of HSP90AA1 protein with the probe.

Diagnosis of recurrence and metastasis of cancer-resistant cancers using the NANOG-dependent resistant cancer diagnostic kit of the present invention comprises the steps of: obtaining a protein sample expressed from a mammalian tissue or cell sample; And identifying and quantifying HSP90AA1 protein in the sample obtained.

Alternatively, the diagnosis of recurrence and metastasis of the anticancer immunity cancer may include obtaining a protein sample expressed from a mammalian tissue or cell sample; And reacting the obtained sample with a monoclonal antibody of HSP90AA1 protein to confirm and quantify the expression of HSP90AA1 protein.

Accordingly, in another aspect, the present invention provides a method for preparing a human body, the method comprising the steps of: (a) measuring the mRNA level or HSP90AA1 protein level of the HSP90AA1 gene from an isolated biological sample; And (b) comparing the measured mRNA level or HSP90AA1 protein level of the HSP90AA1 gene with a control sample, thereby providing information for diagnosing NANOG dependent resistant cancer.

In the present invention, step (b) may be characterized as NANOG dependent resistance cancer, when the mRNA level or HSP90AA1 protein level of the HSP90AA1 gene increases compared to the control.

In addition, the sample is preferably selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid, sweat, urine, ascites fluid and peritoneal fluid, but is not limited thereto.

The measurement method is an ELISA (Enzyme Linked Immunosorbent assay) method for a material (such as protein extracts or plasma of cancer cells or tissues) released from a living body of a diagnostic kit for NANOG dependent resistant cancer containing a monoclonal antibody of HSP90AA1 protein. By quantitative analysis by color reaction or by using HSP90AA1 protein-specific immunostaining (histoimmunostaining) in cancer tissues or cells discharged from the living body can be measured whether the recurrence and metastasis of anti-cancer immunity cancer. Alternatively, by comparing the amount of HSP90AA1 protein expression at the protein level with the normal control group using Western blot analysis on cancer tissues or cells discharged from the living body, information for diagnosis of NANOG dependent resistant cancer may be provided.

Alternatively, providing information for diagnosing the NANOG dependent resistant cancer may include attaching a monoclonal antibody against HSP90AA1 protein to a gel-like support to prepare an immunoaffinity column; Quantifying the HSP90AA1 protein by HPLC using the immunoaffinity column of the above step from a substance (protein extract or plasma of cancer cells or tissues) released from the living body; And comparing and analyzing the quantitative results to determine a degree of recurrence and metastasis of anticancer immune cancer.

The monoclonal antibody of the HSP90AA1 protein may be prepared and used through a monoclonal antibody manufacturing method common in the art, or may be commercially available.

The monoclonal antibody of the HSP90AA1 protein is generally developed using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It can also be quantitatively analyzed by reacting or quantitatively using the conjugated AP or HRP enzyme or the like directly to the HSP90AA1 protein monoclonal antibody.

In addition, a polyclonal antibody that recognizes the HSP90AA1 protein may be used instead of the HSP90AA1 protein monoclonal antibody, which may be manufactured and used through a conventional antiserum manufacturing method in the art.

In addition, the probe may include a PCR primer or nucleic acid probe that specifically binds to the mRNA of HSP90AA1.

In the present invention, the primer for the HSP90AA1 gene is preferably SEQ ID NO: 6 or SEQ ID NO: 7, but is not limited thereto.

RT-PCR or quantitative RT-PCR using primers for the HSP90AA1 gene may be performed to compare the expression level of the HSP90AA1 gene with the normal group to diagnose relapse and metastasis of NANOG dependent resistant cancer.

In addition, Northern blot analysis using the nucleic acid probe for the HSP90AA1 gene may be performed to compare the expression level of NANOG gene with the normal group to diagnose relapse and metastasis of NANOG dependent resistant cancer.

The PCR primer or nucleic acid probe is not particularly limited as long as it is a nucleotide capable of specifically binding to the mRNA of the HSP90AA1 gene.

Reaction and metastasis of the NANOG dependent resistant cancer according to the present invention comprising a primer or nucleic acid probe for detecting the mRNA of the HSP90AA1 gene and a reaction with a substance (RNA extracted from cancer cells or tissues) released from a living body (RT-PCR or quantitatively) RT-PCR or Northern blotting) can be compared to the normal group to perform recurrence and metastasis diagnosis of NANOG dependent resistant cancer.

Diagnosis of relapse and metastasis of the NANOG dependent resistant cancer may include obtaining an RNA sample expressed from a mammalian tissue or cell sample; And reacting the obtained sample with a PCR primer or probe specifically binding to the HSP90AA1 gene to confirm and quantify the expression of the gene.

Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example  1: in human cancer cell lines HSP90AA1  And NANOG  Protein expression confirmation

Human cervical cancer cell lines (SiHa, HeLa, CaSki), colorectal cancer cell lines (HT29, HCT116), lung cancer cell lines (H1299, H3122), melanoma cell lines (A375, 526mel), pancreatic cancer cell lines (Panc-1, cf-pac-1 ) And prostate cancer cell lines (Du145, PC3) in a RIPA buffer (Elpis, Korea) and left for 30 minutes on ice, centrifuged for 30 minutes at 13000rpm in a refrigerated centrifuge, the supernatant is taken to quantify the protein 30㎍ of protein Was loaded onto an SDS-PAGE gel. The gel loaded in PVDF was then transferred and blocked with 5% BSA for 1 hour and then reacted for 12 hours by diluting HSP90AA1 antibody (Thermo Fisher Scientific, USA) and NANOG antibody (Bethyl Laboratories, USA) at 1: 5000. . After washing, the secondary antibody to which HRP was attached was reacted for 1 hour, and then the band of the protein was confirmed by using ECL solution.

As a result, the expression of HSP90AA1 and NANOG protein in various human carcinoma was confirmed, and the correlation between HSP90AA1 and NANOG protein expression could be analyzed (FIG. 1).

Example 2 HSP90AA1 Expression and Patient Survival Rate According to Progression of Cervical Cancer.

Cervical cancer tissue slides were subjected to deparinization using xylene and dehydration using alcohol, and then the slides were immersed in a citrate buffer for 10 minutes in a microwave oven and cooled at room temperature for 1 hour. After washing with distilled water and reacting with 5% goat serum at room temperature for 1 hour, HSP90AA1 antibody (Thermo Fisher Scientific, USA) was diluted 1: 4000 each for 2 hours. After HRP-attached secondary antibody was developed using a DAB kit.

As a result, it was confirmed that the expression of HSP90AA1 increased as the progression of cervical cancer increased in tissues, and the correlation with NANOG expression could be analyzed. In addition, the survival rate of patients with high HSP90AA1 expression was significantly lower in the survival rate obtained through long-term follow-up (FIG. 2).

Example 3 Function of HSP90AA1 in Anticancer Immunity Resistant Cancer Cell Lines

3-1: Confirmation of HSP90AA1 Gene Expression in Anticancer Immunity-resistant Cancer Cell Lines

Total RNA from blast cancer cell line (CaSki P0) and anti-cancer immune resistant cancer cell line (CaSki P3) was isolated using RNeasy Mico Kit (Qiagen) and synthesized cDNA by reverse transcriptase using iScrip cDNA Synthesis Kit (Bio-Rad) It was. Quantitative real-time PCR was performed using iQ SYBR Green super mix (Bio-Rad) and using the CFX96 real time PCR detection protocol. All PCR experiments were repeated three times and quantitative cycle (Cq) values were derived using Bio-Rad CCFX96 Manager 3.0 software. Relative quantification of mRNA levels was calculated by proportional quantification using beta-actin as a control gene. Amplification was performed by PCR using a sense primer (SEQ ID NO: 5'-TGGACAGCAAACATGGAGAG-3 ') and an antisense primer (SEQ ID NO: 7: 5'-AGACAGGAGCGCAGTTTCAT-3') for the HSP90AA1 gene.

3-2: Confirmation of changes in immunity, anticancer and radiation resistance following HSP90AA1 inhibition

Treatment of HSP90AA1-specific siRNA was performed by mixing liposometamine 2000 (lipofectamin 2000, Invitrogene, USA) with a mass ratio of 1: 2 and treating anticancer immune resistant cancer cell lines in a 5% CO ₂ , 37 ° C incubator for 48-72 hours. After incubation, the protein expression of pAKT, MCL1, and CyclinA, including HSP90AA1 and known anticancer immunotolerant NANOG subsignals, was measured.

HSP90AA1-specific siRNAs are shown in SEQ ID NOs: 1-5 in Table 1.

Anti-cancer immunity change experiments were performed using HSP90AA1 expression-inhibited cells obtained in this manner. Carboxyfluorescein succinimidyl ester (CFSE) was used to label CaSki P0 and CaSki P3 cell lines, followed by stimulation of 1ug / ml of E7 peptide (amino acids 49-57, RAHYNIVTF: SEQ ID NO: 8) for 1 hour, followed by E7 specific After mixing CTL (cytotoxic T-lymphocyte) in a 1: 1 ratio, active caspase-3 in CaSki P0 and CaSki P3 cell lines labeled with CFSE was observed through FACS.

As a result, it was confirmed that inhibition of HSP90AA1 expression in anticancer immune resistant CaSki P3 cells increased anticancer immune sensitivity (FIG. 3).

Next, 5000 cells were inoculated into 96-well plates for cell culture using HSP90AA1 suppressed cells. After 12 hours of incubation, 1, 2, 4 uM Cisplatin or 1, 2, 4 Gy radiation was treated for 72 hours, respectively. Each cell viability was measured using the MTT assay.

As a result, it was confirmed that the anti-cancer immune resistant cancer cell line (CaSki P3) had higher sensitivity to Cisplatin and radiation to inhibit HSP90AA1 expression compared to the parent cancer cell line (CaSki P0) (FIG. 3).

3-3: in vitro tumor cell formation

1000 cells of Example 3-2 were placed in DMEM-F12 medium containing 20ng / ml of EGF (Invitrogen, USA), 20ng / ml of bFGF (Invitrogen, USA) and B27 (Gibco, USA). -Super-low adherence plates (Corning, USA) was observed by incubation for 2 weeks. At this time, 100 μl of fresh medium was added every 3 days. The formed tumor cells were quantified by counting tumor spheres of 20-50 μm or more by measuring the diameters of the tumor spheres after obtaining a photographic image using an optical microscope.

As a result, it was confirmed that inhibition of HSP90AA1 expression in tumor-resistant immune CaSki P3 cells reduced tumorigenic capacity (FIG. 3).

Example 4: CaSki Cell Line Expressing Human NANOG

The pMSCV-NANOG vector and liposome preparation (lipofectamin 2000, Invitrogen, USA) prepared in Korean Patent Application Publication No. 10-2012-0083504 were mixed in a mass ratio of 1: 2 to the packaging cell line Phoenix cell (Clontech). Treated. After 24 hours of culture, transformed Phoenix cells were selected with a culture solution containing puromycin (Invitrogen, USA). Selected cells were cultured for 3-4 days to obtain a cell culture medium containing retrovirus, and then concentrated using amicon ultra-15 (millipore, USA). CaSki-NANOG cell line expressing NANOG was prepared by transducing a mixed solution of 500 μl of the final culture concentrate and polybrene (Polybrene, Sigma) to CaSki cell line, a human cervical cancer cell line.

Example 5 Confirmation of Transcription Activity of NANOG Binding to HSP90AA1 Promoter

HEK293 cells were maintained in cell culture medium containing 10% FBS and 105 cells / well were adapted to 12-well plates 1 day prior to analysis. NANOG Wt and NANOG Mut were overexpressed simultaneously with the reporter vector, pGL3-basic, pGL3-HSP90AA1 Wt and GL3-HSP90AA1 Mut. At the same time, all samples were co-transfected with HEK293 cells using lipofectamine 2000 with control vector pCMV-β-Gal to confirm transfection efficiency. After 24 hours, cells were washed with PBS and lysed with Cell Culture Lysis Reagent (Promega, USA). Luciferase activity was measured by Turner Biosystems TD-20 / 20 luminance after addition of 40 μL luciferase assay reagent (Promega, USA). β-galactosidase activity was determined by uQuant microplate reader (BioTek) at 570 nm wavelength after addition of the β-galactosidase measurement reagent containing 1 mM chlorophenol red β-D-galactopyranoside substrate (Roche). , Winooski, VT). Relative luciferase activity was normalized to β-galactosidase activity of cell lysates.

As a result, the expression of HSP90AA1 protein and the expression of HSP90AA1 mRNA increased depending on the transcriptional activity of NANOG, and there was a NANOG binding site in the HSP90AA1 promoter. It is confirmed that the (Fig. 5).

Example 6: Confirmation of Various Treatment Resistance Neutralizes in Vitro by HSP90AA1 Inhibitor, AUY-922

HSP90AA1 in the cervical cancer CaSki cell line overexpressing NANOG, MDA-MB231 P3 cell line, an anti-cancer immunity-resistant breast cancer cell, SiHa, which is naturally overexpressed NANOG, and HCT116 cell line, which is naturally overexpressed colon cancer cell with NANOG. After treatment with the inhibitor, 1 uM of the sublethal dose of AUY-922, it was confirmed whether the AKT signaling mechanism was shared (FIG. 6). At the same time, each cell line confirmed immunity, anticancer and radiation resistance.

Immunoresistant neutralization was confirmed by CaSki P0 and CaSki P3 cell lines using CFSE (Carboxyfluorescein succinimidyl ester), and then 1 ug / ml of E7 peptide (amino acids 49-57, RAHYNIVTF: SEQ ID NO: 8) was stimulated for 1 hour. After E1 specific CTL (cytotoxic T-lymphocyte) was mixed in a 1: 1 ratio, the active caspase-3 in CaSki P0 and CaSki P3 cell lines labeled with CFSE was observed through FACS. .

As a result, it was confirmed that the treatment of AUY-922 increased the anticancer immunity sensitivity in all the various carcinomas in which NANOG is overexpressed including anticancer immunity resistant CaSki P3 and MDA-MB231 P3 cells (FIG. 6).

Next, 5000 cells were inoculated in 96-well plates for cell culture using AUY-922 treated cells. After 12 hours of incubation, 1, 2, 4, 8 uM Cisplatin or 0.5, 1, 2, 4 Gy radiation was treated for 72 hours, respectively. Each cell viability was measured using the MTT assay.

As a result, it was confirmed that the sensitivity of Cisplatin and radiation to AUY-922 treatment was high in various carcinomas overexpressing NANOG, including anticancer immune resistant CaSki P3 and MDA-MB231 P3 cells (FIG. 6).

In addition, each of the 1000 cell lines were placed in DMEM-F12 medium containing 20ng / ml of EGF (Invitrogen, USA), 20ng / ml of bFGF (Invitrogen, USA) and B27 (Gibco, USA). Incubation was performed for two weeks in low adherence plates (Corning, USA). At this time, 100 μl of fresh medium was added every 3 days. The formed tumor cells were quantified by counting tumor spheres of 20-50 μm or more by measuring the diameters of the tumor spheres after obtaining a photographic image using an optical microscope.

As a result, it was confirmed that AUY-922 treatment reduced tumor cell formation ability in various carcinomas in which NANOG was overexpressed, including anticancer immune resistant CaSki P3 and MDA-MB231 P3 cells (FIG. 6).

Example  7: HSP90AA1  Inhibitors, AUY By -922 In vivo  Confirmation of neutralization of anticancer immunity

MDA-MB231 P3 cells were injected 2 x 10 ⁶ into the ventral epidermis of each NOD-SCID mouse group (female, 5 weeks old, n = 5, central experimental animals) and AUY-922 (0.05 mg / kg) on day 7 ) Was injected by intraperitoneal injection. At this time, the control was confirmed by injecting the same volume of PBS. 2 x 10 ⁶ MART-1-specific T cells were immunized intravenously with IL-2 the next day after inhibitor injection. The same set was run at 7 day intervals and repeated 3 times. For each mouse, the tumor volume was measured by caliber (Mitutoyo, Japan), and the tumor volume was calculated by the formula of (short axis x long axis 2) / 2.

As a result, it was confirmed that tumor formation was reduced as a result of the combination of HSP90AA1 inhibitory compound and immunotherapy using CD8 + T cells (FIG. 7).

In addition, Figure 7 confirms that the inhibitory effect is increased by the inhibitor AUY-922 of HSP90AA1 in the immunotherapy model for human cancer cells. AUY-922 uses commercially available drugs and Luminespib purchased from selleckchem.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Use of HSP90AA1 for Treating or Diagnosing NANOG-mediated          Diseases <130> P17-B215 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA 1 <400> 1 caccaaacat aacgatgatg a 21 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> siRNA 2 <400> 2 cagttggaat tcagagccct tctat 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> siRNA 3 <400> 3 agttggaatt cagagccctt ctatt 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> siRNA 4 <400> 4 acagttggaa ttcagagccc ttcta 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> siRNA 5 <400> 5 aggacagttg gaattcagag ccctt 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S primer <400> 6 tggacagcaa acatggagag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS primer <400> 7 agacaggagc gcagtttcat 20 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> E7 peptide <400> 8 Arg Ala His Tyr Asn Ile Val Thr Phe   1 5

Claims (12)

A composition for preventing or treating NANOG dependent resistant cancer containing HSP90AA1 (heat shock protein AA1) as an active ingredient.
The composition of claim 1, wherein the expression or activity inhibitor is an antibody, aptamer or compound that specifically binds to siRNA or HSP90AA1 protein for HSP90AA1 gene.
The composition of claim 2, wherein the compound is AUY-922.
The composition according to claim 1, which is used in combination with an immunotherapeutic agent.
The composition of claim 1, wherein the resistant cancer is an anticancer agent or an anticancer immune cancer.
A diagnostic kit for NANOG dependent resistant cancer, comprising a probe capable of amplifying the HSP90AA1 gene or a probe capable of hybridizing under HSP90AA1 gene.
The kit of claim 6, wherein the resistant cancer is an anticancer agent or an anticancer immune resistant cancer.
How to provide information for diagnosis of NANOG dependent resistant cancer, including the following steps:
(a) measuring the mRNA level or HSP90AA1 protein level of the HSP90AA1 gene from the isolated biological sample; And
(b) comparing the measured mRNA level or HSP90AA1 protein level of the HSP90AA1 gene with a control sample.
The method of claim 8, wherein the step (b) is determined as NANOG dependent resistant cancer when the mRNA level or HSP90AA1 protein level of the HSP90AA1 gene is increased compared to the control group.
The method of claim 8, wherein the resistant cancer is an anticancer agent or an anticancer immune resistant cancer.
The method of claim 8, wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid, sweat, urine, ascites fluid and peritoneal fluid.
According to claim 8, The measurement is immunoblot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip Method for providing information, characterized in that using any one selected from the group consisting of.

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