WO2013122321A1 - Utilisation de hdac6 en tant que marqueur d'hépatome et agent thérapeutique - Google Patents

Utilisation de hdac6 en tant que marqueur d'hépatome et agent thérapeutique Download PDF

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WO2013122321A1
WO2013122321A1 PCT/KR2012/011549 KR2012011549W WO2013122321A1 WO 2013122321 A1 WO2013122321 A1 WO 2013122321A1 KR 2012011549 W KR2012011549 W KR 2012011549W WO 2013122321 A1 WO2013122321 A1 WO 2013122321A1
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hdac6
liver cancer
protein
gene
expression
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남석우
정광화
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가톨릭대학교 산학협력단
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

  • HDAC 6 is a Marker for Diagnosing Liver Cancer and as a Therapeutic Agent
  • the present invention relates to a novel liver cancer diagnostic marker that can effectively diagnose and predict liver cancer early, a liver cancer diagnostic kit, a method for diagnosing liver cancer using the liver cancer diagnostic marker, a composition for treating liver cancer using the marker substance, and a screening method thereof. All-
  • Liver cancer is the fifth most common cancer in the world, but the resulting mortality is the third most aggressive cancer (Ahn J, Flamm SL. Hepatocellular carcinoma. Dis Mon 2004; 50: 556-573).
  • Therapeutic surgery is only possible in 15% to 25% of patients, and most liver cancer patients die in a relatively short time due to locally advanced or metastatic disease (Roberts LR, Gores GJ. Hepatocellular carcinoma: molecular pathways and new therapeutic targets.Semin Liver Dis 2005; 25: 212-225).
  • Hepatitis B virus, hepatitis C virus, and aflatoxin BKaflatoxin B1 are known as major causes of liver cancer.
  • HDACs Histone deacetylases
  • corepressors or multi-protein transcriptional complexes where they do not bind DNA directly but instead bind chromatin. Regulate transcription through transformation (Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF.Histone deacetylases: unique players in shaping the epigenetic histone code.Ann NY Acad Sci 2003; 983: 84-100 ).
  • HDAC11 human HDACs
  • HDAC11 class IV enzyme
  • HDACs pathological activity and deregulation of HDACs can cause various diseases such as cancer, immune disease, and muscular dystrophy (Yang XJ, Seto).
  • HATs and HDACs from structure, function and regulation to novel strategies for therapy and prevention.Oncogene 2007; 26: 5310-5318).
  • HDACs the specific roles played by individual HDACs in the regulation of cancer development are not yet known.
  • HDAC6 is a member of the class lib family of HDACs, acts as a cytoplasmic deacetylase associated with microtubules (MTs), and deacetylates alpha-tubulin. (Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al. HDAC6 is a microtubule-associated Misl8a. Nature 2002; 417: 455-458).
  • Microtubule-related HDAC6 is a key component in the lysosomal protein breakdown pathway and directly reacts with misfolded or poly-ubiquitated proteins to prevent aggresome ( Aggresome formation / autophagy leads to lysosomal-mediated protein degradation (Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao TP.The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 2003; 115: 727-738, Boyault C, Zhang Y, Fritah S, Caron C, Gilquin B, Kwon SH, et al.
  • HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev 2007; 21: 2172-2181. Recently, it has been shown that HDAC6 regulates autophagosome maturation and is involved in autophagy regulation in the autophagosome-lysosomal fusion phase (Lee JY, Koga H, Kawaguchi Y, Tang W, Wong) E, Gao YS, et al. HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. EMBO J; 29: 969-980).
  • HDAC6 plays a role in the transport of misfolded proteins during aggresome proteolysis, thereby protecting cells from the harmful effects of protein coagulation (Kwon S, Zhang Y, Matthias P).
  • the deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response.Genes Dev 2007; 21: 3381-3394).
  • HDAC6 has also been reported to regulate epithelial-mesenchymal transition in several cancers through the regulation of several key cellular functions (Lee YS, Lim KH, Guo X, Kawaguchi Y, Gao Y).
  • HDAC6 for transforming growth factor—betatal induced epithelial ⁇ mesenchymal transition.J Biol Chem 2008; 283: 21065-21073), expression of HDAC6 results in oncogenic transformation, anchorage-independent proliferation, and tumor There is a lot of evidence to support the fact that it is related to tumor aggressiveness.
  • HDAC6 up-regulation of HDAC6 in various tumors and cell lines has shown that HDAC6 will play an important role in cancer.
  • HDAC6 showed down-regulation in liver cancer compared to normal tissue, and according to the initial analysis of HDAC6 in human liver cancer tissue, It was confirmed that expression was reduced. Therefore, the present inventors measured HDAC6 expression in tumors and normal tissues using a tissue microarray (TMA) assay to elucidate the biological function of HDAC6 as a tumor suppressor in liver tumorigenesis.
  • TMA tissue microarray
  • the present invention has been completed. Tumor inhibitory activity of HDAC6 was also confirmed by in vivo experiments using cell lines stably overexpressing HDAC6.
  • An object of the present invention is to provide a use of the HDAC6 (Histone deacetylase 6) gene or a protein encoded from the gene as a marker for diagnosing liver cancer.
  • HDAC6 Histone deacetylase 6
  • another object of the present invention is to provide a method for predicting and diagnosing liver cancer comprising measuring the amount of HDAC6 gene expression or protein.
  • Another object of the present invention is to provide a method for screening a substance capable of preventing or treating liver cancer.
  • Another object of the present invention to provide a composition for treating liver cancer using HDAC6.
  • the present invention provides a marker for diagnosing liver cancer consisting of HDAC6 (Histone deacetylase 6) gene or HDAC6 protein encoded from the gene.
  • HDAC6 Histone deacetylase 6
  • the HDAC6 gene may be one having a nucleotide sequence of SEQ ID NO: 1.
  • the present invention provides a kit for diagnosing liver cancer comprising a substance for measuring the level of HDAC6 (Histone deacetylase 6) gene or the level of HDAC6 protein.
  • the material for measuring the level of the HDAC6 gene may be a primer or probe that can amplify the HDAC6 gene.
  • the substance for measuring the level of the HDAC6 protein may be an antibody that specifically recognizes the HDAC6 protein.
  • the present invention comprises the steps of (a) measuring the amount of expression or protein amount of HDAC6 (Histone deacetylase 6) gene present in a biological sample; And (b) provides a method for predicting and diagnosing liver cancer comprising the step of comparing the measurement results of the step (a) with the amount of expression or protein amount of the HDAC6 gene of the control sample.
  • HDAC6 Histone deacetylase 6
  • the biological sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.
  • the method for measuring the expression amount of the HDAC6 gene or the amount of HDAC6 protein but is not limited thereto, reverse transcriptase-polymerase chain reaction, Real time one polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), immunoblot analysis, immunohistochemical staining, radioimmunoassay (RIA), It may be selected from the group consisting of radioimmunodif fusion and immunoprecipitation assay.
  • the present invention (a) contacting a sample to be analyzed to liver cancer cells containing HDAC6 (Histone deacetylase 6) gene or HDAC6 protein; (b) measuring the expression level of the HDAC6 gene, the amount of HDAC6 protein, or the activity of HDAC6 protein Doing; And (c) determining that the sample is a material for preventing or treating liver cancer when the measurement result of step (b) increases the expression level of HDAC6 gene, the amount of HDAC6 protein, or the activity of HDAC6 protein.
  • HDAC6 Histone deacetylase 6
  • the method for measuring the expression amount of the HDAC6 gene, the amount of HDAC6 protein or the activity of the HDAC6 protein is not limited thereto, reverse transcriptase-polymerase chain reaction ), real-time polymerase chain banung (real time-polymerase chain reaction), Western blot, Northern blot, ELISA (enzyme linked immunosorbent assay), immune, tablet: (immunoblot) analysis, the surface station staining, radioimmunoassay analysis ( RIA: may be selected from the group consisting of radioimmunoassay, radioimmunodif fusion, and immunoprecipitation assay.
  • the present invention includes HDAC6 (Histone deacetylase 6) or a substance that promotes its expression, and activates the JNK-mediated Beclin 1 pathway to prevent cell death by autophagy It provides a composition for treating liver cancer, characterized by having a mechanism to promote.
  • HDAC6 Histone deacetylase 6
  • a substance that promotes its expression and activates the JNK-mediated Beclin 1 pathway to prevent cell death by autophagy It provides a composition for treating liver cancer, characterized by having a mechanism to promote.
  • HDAC6 gene according to the present invention was confirmed that the amount of expression in the tissues or cells of liver cancer compared to normal tissues or cells. Therefore, when the HDAC6 gene is used as a marker for diagnosing liver cancer, liver cancer can be rapidly and accurately diagnosed and predicted early, and further, it can be used as a target for the development of liver cancer therapeutics.
  • HDAC6 expression in human liver cancer tissues and liver cancer cell lines shows downregulation or elimination of HDAC6 expression in human liver cancer tissues and liver cancer cell lines.
  • A Gene expression of HDAC6 mRNA based on microarray analysis of preneoplastic nodules.
  • LGDN low-grade dysplastic nodule
  • HGDN highgrade dysplastic nodule
  • Gl-3 Edmondson grades ⁇
  • B HDAC6 expression in human liver cancer and adjacent non-tumor tissues was analyzed by Western blot.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • C Immunohistochemical staining for HDAC6 in human liver cancer and adjacent non-tumor tissue pairs loaded into tissue microarrays.
  • D Endogenous HDAC6 expression in human liver cancer cell lines by Northern and Western blot analysis. Beta-actin probes and GAPDH were used to regulate RNA and protein loading, respectively. Each experiment was performed at least twice.
  • FIG. 2 shows that topic expression of HDAC6 in Hep3B cells induces caspase-independent : apoptosis.
  • A Cells were transduced with control (None), empty vector (Mock), or lg HDAC6 expression vector (pcDNA ⁇ HDAC6).
  • the left panel measures the levels of HDAC6 and acetylated alpha leubulin (Ac ⁇ ⁇ -tubulin) by Western blot analysis. Alpha-tubulin was used as loading control.
  • the right panel shows growth for a control (Mock or None) of Hep3B cells overexpressing HDAC6. Cell counts were determined by nuclear staining with trypan blue. Data is expressed as mean and standard deviation of three independent experiments.
  • C Flow cytometry results of Hep3B cells transduced with control (None), empty vector (Mock), or 1 or 2 HDAC6 expression vector (pcDNA_HDAC6). FITC-labeled Annexin V-positive cells (upper right and lower right) were considered apoptotic cells. Two independent experiments were performed.
  • FIG. 3 shows that topic expression of HDAC6 in Hep3B cells induces apoptosis by autophagy.
  • A Cells were transduced with a control (None), empty vector (Mock), or 1 or 2 HDAC6 expression vector (pcDNA_HDAC6).
  • the left panel is a Western blot analysis of the levels of HDAC6, LC3B-II, and acetylated alpha-leuulin (Ac- ⁇ —tubulin).
  • the right panel shows the alpha-leubulin to LC3B-II ratio measured by densitometric analysis.
  • HDAC6 induces apoptosis by autophagy in liver cancer cell lines.
  • HDAC6 overexpressing cell lines were established as described in the Examples of the present invention. Left panel, immunoblot analysis shows persistent HDAC6 overexpression, which is confirmed by hypoacetylation of alpha-tubulin in HDAC6 overexpressing cell lines (Hep3B_HDAC6 clone # 1 and clone # 2) versus control (Hep3B_Mock) . Right panel, cell growth analysis of established HDAC6 overexpressing cell lines (Hep3B_Mock, Hep3B_HDAC6 clones # 1 and # 2). Relative growth was measured via the MTT assay.
  • FIG. 6 shows that HDAC6 overexpression activates autophagy by upregulating Beclin 1 expression.
  • Beclin 1 expression was examined by Western blot analysis in control (Hep3B_Mock) and HDAC6 overexpressing cell lines (Hep3B_HDAC6 clones # 1 and # 2). Hep3B cells treated with 20 uM ceramide for 24 hours were used as a positive control for Beclin 1 transduction.
  • graph bar shows LC3B-II to ⁇ -tubulin ratio (unpaired student's t test, * P ⁇ 0.05 vs. Mock).
  • HDAC6 overexpressing cells Hep3B_HDAC6 clone # 1 were transfected with control (None), 100 nM scrambled siRNA (Scr), or 100 nM or 200 nM HDAC6-specific siRNA. Thereafter, Beclin 1 and LC3B-II expression levels were measured by Western blot analysis.
  • graph bar shows Beclin 1 to a-tubulin ratio (unpaired student's t test, * P ⁇ 0.05 vs. Scr).
  • FIG. 7 shows JNK / c-Jun activity by HDAC6 mediated Beclin 1′-dependent autophages in liver cancer cells.
  • A JNK, phospho— JNK (p-JNK), c-Jun and the control (Hep3B_Mock) and HDAC6 overexpressing cell lines (Hep3B_HDAC6 Clone # 1 and # 2) The expression level of phospho-c-Jun (p— c-Jun) was measured by immunoblotting.
  • B HDAC6 overexpressing cells (Hep3B ⁇ HDAC6 Clone # 1) were transduced with control (None), 100 nM scrambled siRNA (Scr), or 50 nM or 100 nM HDAC6-specific siRNA.
  • HDAC6 transduced with specific siRNA followed by immunoblotting HDAC6, JNK, phospho-JNK (p-JNK), c-Jun, phospho— c— Jun (pc-Jun), Beclin 1, and LC3B-II
  • C HDAC6, JNK, phospho-JNK (p-JNK), with or without 10 uM SP600125 in control (Hep3B_Mock) and HDAC6 overexpressing cell clones (Hep3B_HDAC6 Clone # 1) to inhibit JNK signaling
  • Expression levels of c-Jun, phospho-c-Jun (pc-Jun), Beclin 1 and LC3B-II proteins were measured by immunoblotting. All membrane blots were reprobed for alpha-leubulin to confirm balanced loading.
  • HDAC6 shows the effect of HDAC6 on ROS accumulation.
  • Hep3B cells were either untreated (None), or transduced for 48 hours with empty mock vectors (Mock), 1 and 2 / g of pcDNA 3.1-HDAC6 expression vector (pcDNA_HDAC6). Cells were then stained with H2DCFDA for 30 minutes and cell ROS levels were observed by flow cytometry.
  • B Graph bars indicate relative levels of ROS compared to control (None).
  • HDAC6 a cytoplasmic deacetylase
  • HDAC6 can function as a tumor suppressor in human liver cancer cells by mediating cell death by caspase-independent autophagy via a dependent pathway of JNK-activated Beclin 1. Said that. Expression of HDAC6 is inhibited or lost in liver cancer, and according to an embodiment of the present invention, ectopic expression of HDAC6 inhibited tumor growth in both in vitro and in vivo experiments. In addition, HDAC6 activated JNK / cJ Jun signaling pathway in liver cancer cells to activate Beclin 1 / LC3B-II dependent autophagy.
  • HDAC6 plays an important role in the pathogenesis of liver cancer, suggesting that HDAC6 may have important therapeutic effects in the treatment of liver cancer.
  • Acetylation of histones by lysine is a major epigenetic regulator of chromatin conformation and gene expression.
  • the dynamic nature of histone acetylation is determined by the balance between histone acetyltransferase activity and HDAC enzymes (Thiagalingam S, et al. Histone deacetylases: unique players in shaping the epigenetic histone code. Ann NY Acad Sci 2003; 983: 84-100).
  • HDACs are involved in a variety of cellular activities such as gene transcription, protein expression, protein-protein interactions, protein intracellular localizations, and cellular signaling (Witt O, et al. HDAC family: What are the cancer relevant targets? Cancer Lett 2009; 277: 8-21).
  • HDACs regulate the expression and activity of several proteins associated with the development and progression of cancer (Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK.Histone deacetylases and cancer: causes and therapies.Nat Rev Cancer 2001; 1: 194-202, and Glozak MA, Seto E. Histone deacetylases and cancer.Oncogene 2007; 26: 5420-5432).
  • HDAC family members are aberrantly expressed in some tumors and have a non-incidental function of regulating the signs of cancer cells (Witt 0, et al, 2009 and Marks P, et al, 2001). Since abnormal HDAC activity was initiated during the tumorigenization process, considerable effort has been put into developing HDAC inhibitors that can modify histone acetylation status and promote the re-expression of tumor suppressor genes that have stopped abnormal expression. It was.
  • HDAC6 is overexpressed in many human cancer cell lines and mouse tumor models.
  • HDAC6 has been regarded as a therapeutic target because cancer cells play an important role in many of the signaling pathways necessary for survival and appearance (Aldanai Masangkay GI, Sakamoto KM.The role of HDAC6 in cancer J Biomed Biotechnol; 2011: 875824).
  • HDAC6 expression can be expressed in squamous cell cancer (Sakuma T, et al. Aberrant expression of histone deacetylase 6 in oral squamous cell carcinoma. Int J Oncol 2006; 29: 117-124), human breast cancer tissues ( Yoshida N, et al.
  • HDAC6 has anti-tumor function in liver cancer production process.
  • the present inventors found that the ectopic expression of HDAC6 inhibits the growth and proliferation of liver cancer cells without affecting cell cycle progression or apoptosis (FIG. 2).
  • the above findings suggest that HDAC6 acts as a tumor suppressor in the development of liver cancer, and thus is contrary to previous reports on the tumorigenic function of HDAC6 in the development and progression of cancer.
  • HDAC6 is located only in the cytoplasm that can be associated with microtubules and actin cytoskeleton. In addition, HDAC6 is believed to play an important role in the intracellular management of protein pools. Unlike other HDAC family members, HDAC6 has ubiquitin ⁇ binding activity and is associated with both microtubules and F ⁇ actin cytoskeleton (Hubbert C, et al. HDAC6 is a microtubule associated associated deacetylase. Nature 2002; 417: 455 -458 and Zhang X, Yuan Z, Zhang Y, Yong S, Salas' Burgos A, Koomen J, et al.
  • HDAC6 modulates cell motility by altering the acetylation level of cortactin.Mol Cell 2007; 27: 19 he 213). Recently, it has been reported that HDAC6 regulates the fusion of autophagosomes and lysosomes, an evolutionarily conserved protein-degradation pathway in eukaryotes, and regulates autophagy, which is essential for cell survival in nutrient-restricted conditions.
  • Lee JY, et al. HDAC6 controls autophagosome maturation essential for ubiquitin— selective quality-control autophagy. EMBO J; 29: 969-980 and Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy.
  • Autophagy has a variety of pathways, and abnormalities in the autophagy signaling pathway have been found in cancer and many other diseases. Paradoxically, autophagy has been reported to promote cell death in the course of cell survival and cancer development and in cancer treatment (RosenfeWt MT, Ryan KM. The multiple roles of autophagy in cancer. Carcinogenesis; 32: 955—963). , Bonn. Examples of the invention show that HDAC6 functions as a tumor suppressor agent by activating apoptosis by autophagy in liver cancer.
  • mTOR mimalian target of rapamycin
  • AIF apoptosis-inducing factor
  • ROS reactive oxygen species
  • CDK cyclin-dependent kinase
  • Beclin 1 overexpresses HDAC6 in mouse tumor tissue. Induced from Hep3B cells (FIG. 5F).
  • Beclin 1 may play an important role in binding proteins from the cell to the autophagy or providing an autophagy pathway to membrane constructs (Liang XH, et al. Induction of autophagy and inhibition of tumorigenesis by beclin Nature 1999; 402: 672-676), and can also be an important 'molecular switch' of autophagy and apoptosis in mammals (Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis.Cell Death Differ; 18: 571 ⁇ 580). In addition, Beclin 1 dysfunction has been observed in various diseases including cancer, and therefore, it is thought to be able to function as a tumor suppressor by promoting macroautophagy of cells.
  • HDAC6 overexpression in Hep3B cells promoted Beclin 1 expression in a manner similar to ceramide, a potential autophagy inducer.
  • siRNA silencing of HDAC6 blocked Beclin 1 induction and autophagy, which was confirmed by reduced LC3B-II conversion (FIGS. 6A and B).
  • the link between Beclin 1 induction and HDAC6 ⁇ induced autophagy was further enhanced by the fact that 3—MA inhibited Beclin 1 expression in HDAC6 overexpressing cells (FIG. 6C).
  • the present inventors focused on the relationship between JNK pathway activity and Beclin 1 expression in HDAC6-induced autophagy.
  • the JNK pathway is activated during HDAC6—induced autophagy in liver cancer cell lines, which has been shown to induce Beclin 1 expression.
  • siRNAs targeting the JNK-specific inhibitors SP600125 and HDAC6 inhibited Bedin 1 up-regulation and autophagy induction (FIGS. 6B and 7C).
  • c-Jun was also shown to be related to Beclin 1 regulation of HDAC6—induced autophagy (FIG. 7).
  • the present invention provides a method of inhibiting or inhibiting HDAC6 expression in liver cancer; Efficacy expression of HDAC6 shows that it inhibits tumor growth in vitro and in vivo by promoting autophagy apoptosis and activating the JNK-mediated Beclin 1 pathway. It demonstrates that it functions as a tumor suppressor by activating caspase—independent autophagy apoptosis, thus suggesting the clinical potential of HDAC6 as a therapeutic agent for liver cancer. Accordingly, the present invention provides a marker for diagnosing liver cancer comprising an HDAC6 (Histone deacetylase 6) gene or an HDAC6 protein encoded from the gene.
  • HDAC6 Histone deacetylase 6
  • diagnosis refers to confirming a pathological condition.
  • diagnosis means confirming the presence or absence of expression of a liver cancer diagnostic marker and confirming the occurrence of liver cancer.
  • Diagnosis in the present invention includes determining whether liver cancer diagnostic markers are expressed and the degree of expression, and determining whether the liver cancer is developed, developed, and alleviated.
  • the diagnostic marker refers to a substance capable of distinguishing the cells of liver cancer from the normal cells and diagnosed, a polypeptide or nucleic acid (for example, mRNA that shows an increase or decrease in cells of liver cancer compared to normal cells) Organic lipid molecules, such as lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides), and the like.
  • the marker for diagnosing liver cancer provided by the present invention may be an HDAC6 gene or a protein whose expression amount is reduced in cells of liver cancer compared to normal cells, and preferably, the HDAC6 gene has a nucleotide sequence set forth in SEQ ID NO: 1.
  • the present invention provides a composition for diagnosing liver cancer comprising a substance for measuring the level of HDAC6 gene or the level of HDAC6 protein.
  • the level of the HDAC6 gene preferably, the mRNA level of the HDAC6 gene is expressed, that is, the amount of mRNA, and the substance capable of measuring the level may include a primer or probe specific for the HDAC6 gene.
  • the primer or probe specific for the HDAC6 gene may be a primer or probe capable of specifically amplifying the entire HDAC6 gene represented by SEQ ID NO: 1 or a specific region of the gene, and the primer or probe may be You can design through the public way.
  • primer refers to a single that can serve as an initiation point for template—indicative DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature and in a complete bedrock. -Refers to stranded oligonucleotides. Suitable lengths of primers can vary depending on the use of various factors such as silver and prima. In addition, the sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and may be divided if it has a broad complementarity within a range capable of being unique with the template to allow the primer to function uniquely.
  • the primers of the present invention do not need to have a sequence that is perfectly complementary to the nucleotide sequence of the HDAC6 gene, which is a template, and is sufficient to have a sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. .
  • the primer according to the present invention can be used for gene amplification reaction.
  • the amplification reaction refers to reactions that amplify nucleic acid molecules, and the amplification reactions of such genes are well known in the art, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Ligase chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • LCR Ligase chain reaction
  • TMA electron mediated amplification
  • NASBA nucleic acid sequence substrate amplification
  • the term probra is a natural or modified monomer or chain It refers to a linear oligomer of (linkages), including deoxyribonucleotides and ribonucleotides, and can specifically hybridize to the target nucleotide sequence, present naturally or artificially synthesized.
  • the probe according to the invention may be a single chain, preferably oligodioxyribonucleotides.
  • Probes of the invention may include natural dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives.
  • the probes of the present invention may also comprise ribonucleotides.
  • the probes of the present invention may be backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, flax-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha -DNA And methylphosphonate DNA, sugar modified nucleotides such as 2 ' ⁇ O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'- O-allyl DNA, 2′-0—alkynyl DNA, nuclear source DNA, pyranosyl RNA and anhydronucleophilic DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are MO—, Chloro-, Iodo-, Methyl ⁇ ,
  • the substance capable of measuring the level of the HDAC6 protein in the present invention may include a polyclonal antibody, a monoclonal antibody and a recombinant antibody round antibody that can specifically bind to the HDAC6 protein.
  • a method for generating an antibody using the protein may be known by a person skilled in the art. It can be manufactured easily.
  • HDAC6 antigen can be injected into an animal and collected from the animal to obtain a serum containing the antibody, and the polyclonal antibody can be produced by a method well known in the art.
  • the hybridoma method is well known in the art. Can be prepared using the method (Kohler et al, European Jounral of Immunology, 6, 511-519, 1976) or phage antibody library (Clackson et al, Nature, 352, 624-628, 1991, Marks et al, J Mol. Biol., 222: 58, 1-597, 1991).
  • the antibodies according to the invention may comprise not only a complete form having two full length light chains and two full length heavy chains, but also functional fragments of antibody molecules.
  • a functional fragment of an antibody molecule refers to a fragment that retains at least antigen binding functions and includes Fab, F (ab '), F (ab') 2 and Fv rounds.
  • the present invention also provides a kit for diagnosing liver cancer comprising a composition for diagnosing liver cancer capable of measuring the expression level of an HDAC6 protein or a gene encoding the same.
  • Liver cancer diagnostic composition included in the liver cancer diagnostic kit of the present invention may include a primer, a probe or an antibody that can measure the expression level of the HDAC6 protein or a gene encoding the same, the definition thereof is as described above.
  • the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as complete fluid, DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus ( Tth), Thermus filif ormis, Thermis flavus, Thermococcus liter alis or Pyrococcus to furiosus (Pfu) '' a thermal stability resulting from DNA jeunghap enzyme), may include a DNA polymerase joinja and dNTPs, in the invention of liver cancer diagnostic kit
  • the kit of the present invention may optionally comprise a secondary antibody and a substrate of the label.
  • the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.
  • the present invention provides a liver cancer diagnostic microarray comprising a composition for diagnosing liver cancer capable of measuring the expression level of HDAC6 protein or a gene encoding the same.
  • a primer, probe or antibody capable of measuring the expression level of an HDAC6 protein or gene encoding it is used as a hybridizable array element and immobilized on a substrate:
  • Preferred substrates are suitable rigid or semi-rigid supports, for example membranes, filters, chips, Slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by chemical bonding methods or covalent binding methods such as UV.
  • the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coated surface.
  • the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).
  • sample to be applied to the microarray of the present invention is a nucleic acid label
  • the present invention can provide a method for diagnosing liver cancer by measuring the expression level or HDAC6 protein level of the HDAC6 gene, the method (a) the amount of expression of the HDAC6 (Histone deacetylase 6) gene present in the biological sample or Measuring the amount of protein; And (b) comparing the measurement result of step (a) with the expression amount or protein amount of the HDAC6 gene of the control sample.
  • the method of measuring the expression level or HDAC6 protein level of the HDAC6 gene in the above may be carried out including a known process for separating mRNA or protein from biological samples using known techniques.
  • the "biological sample” refers to a sample collected from a living body, which is different from the normal control, in which the expression level or protein level of the HDAC6 gene according to the liver cancer occurrence or progression is used.
  • it may include tissue, cells, blood, serum, plasma, saliva and urine.
  • the expression level of the HDAC6 gene is preferably to measure the level of mRNA, the method of measuring the level of mRNA reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blots and DNA chips, but are not limited thereto.
  • the HDAC6 protein level may be measured using an antibody.
  • the HDAC6 marker protein and a specific antibody in the biological sample form a conjugate, that is, an antigen-antibody complex, and the amount of the antigen-antibody complex is detected. Quantitative measurements can be made through the magnitude of the signal on the label.
  • the detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not limited thereto.
  • the present invention can determine the amount of HDAC6 mRNA expression or protein in the control group and the amount of HDAC6 mRNA expression or protein in liver cancer patients or suspected liver cancer patients through the above-described detection methods, and the degree of expression By comparing with, we can predict and diagnose the development, progression or prognosis of liver cancer.
  • the present invention further comprises the steps of: (a) contacting a sample to be analyzed with a liver cancer cell comprising a HDAC6 (Histone deacetylase 6) gene or an HDAC6 protein; (b) measuring the expression amount of the HDAC6 gene, the amount of HDAC6 protein or the activity of HDAC6 protein; And (c) determining that the sample is a material for preventing or treating liver cancer when the measurement result of step (b) increases the expression level of HDAC6 gene, the amount of HDAC6 protein, or the activity of HDAC6 protein.
  • a method for screening a substance for preventing or treating liver cancer is provided.
  • a sample to be analyzed may be contacted with liver cancer cells containing the HDAC6 gene or protein.
  • the sample refers to an unknown substance used in screening to examine whether the expression of HDAC6 gene, the amount of HDAC6 protein, or the activity of HDAC6 protein is affected.
  • the sample may include, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.
  • the expression level of the HDAC6 gene, the amount of HDAC6 protein or the activity of the HDAC6 protein can be measured in the cells treated with the sample.
  • the sample may be determined as a substance capable of treating or preventing liver cancer.
  • the method of measuring the expression amount of HDAC6 gene, the amount of HDAC6 protein or the activity of HDAC6 protein in the above can be carried out through various methods known in the art, for example, but not limited to reverse transcriptase polymerase Reverse transcriptase-polymerase chain reaction, real time—polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay, immunoblot analysis, immunohistochemical staining , Radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assays.
  • the present 'invention provides a composition for treating liver cancer comprising a gene or HDAC6 protein HDAC6 (Histone deacetylase 6) as an active ingredient.
  • the HDAC6 gene is inserted into an animal cell expression vector.
  • the HDAC6 protein is characterized in that it has a mechanism for promoting cell death by autophagy by activating the JNK-mediated Beclin 1 pathway.
  • the composition contains the HDAC6 gene or HDAC6 protein as an active ingredient for use as a pharmaceutical composition.
  • the HDAC6 gene included in the pharmaceutical composition of the present invention is inserted into an expression vector such as a plasmid or a viral vector, and then introduced into the target cell by a method known in the art, such as transfectic. Can be used for gene therapy.
  • a gene delivery method using a plasmid expression vector is a method for delivering plasmid DNA directly to human cells, which can be used by a person approved by the FDA (Nabel, E. G., et al., Science, 249: 1285-1288, 1990).
  • Plasmid DNA has the advantage of being able to be homogeneously purified, unlike viral vectors.
  • mammalian expression plasmids known in the art can be used.
  • mammalian expression plasmids known in the art can be used.
  • pRK5 European Patent No. 307,247
  • pSV16B Counter Patent Publication No. 91/08291
  • pVL1392 PharMingen
  • pCDNA3.1 Invitrogen
  • a plasmid expression vector comprising a nucleic acid according to the present invention is a method known in the art, for example, but not limited to, transient transfection, microinjection, transduction ), Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection transfection, electroporation, gene guns and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).
  • known suitable viral vectors can be used in the pharmaceutical compositions of the present invention.
  • a cell line stably overexpressing HDAC6 was produced through liposome-mediated transfection.
  • the HDAC6 expression vector synthesizes the full-length HDAC6 coding sequence insert that can express the HDAC6 protein by PCR and inserts it into the pcDNA 3,1 (invitrogen) vector to produce the HDAC6_pcDNA 3,1 vector. It was.
  • HDAC6 ⁇ pcDNA 3,1 vector was transiently transfected using liposome (lipofectamine 2000, invitrogen) to Hep3B liver cancer cell line where HDAC6 expression was inhibited, and in fact, HDAC6 overexpressed in Hep3B and various liver cancer cell lines. It was confirmed that the inhibition of cell growth. This phenomenon was thought to be caused by the overexpression of HDAC6 caused by autophagic cell death in liver cancer. Then, to investigate the tumor suppression effect of HDAC6 in liver cancer suppression in animal experiments, cells that stably overexpress HDAC6 were prepared. The method is as follows. The HDAC6_pcDNA 3,1 vector is transiently transfected into liver cancer cells.
  • liver cancer cells are selected using geneticin (antibiotic). Since HDAC6_pcDNA 3,1 vector is resistant to geneticin, cells containing HDAC6 ⁇ pcDNA 3,1 vector will continue to grow without geneticin killing. Single cells containing HDAC6NA pcDNA 3,1 vector continue to grow and call When one colony is extracted and each cell is grown again, one cell becomes a clone in which HDAC6 is stably overexpressed. After the production of the HDAC6 overexpressing cells, it was confirmed that the effect of transfection with the HDAC6 transient, and then the animal experiments. Finally, HDAC6 siRNA (Dharmacon) transient transfection was performed to stably overexpressed cells to determine whether autophagic cell death caused by HDAC6 overexpression was suppressed again.
  • HDAC6 siRNA Dharmacon
  • the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc. when administered to humans. Examples include stable oral carriers such as lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid and parenteral administration carriers such as water, suitable oils, saline, aqueous glucose and glycol dung.
  • suitable preservatives include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid
  • suitable preservatives include benzalkonium chloride, methyl- or propyl—para Ben and chlorobutanol
  • Other pharmaceutically acceptable carriers include Reference may be made to Hunt (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995.)
  • the pharmaceutical composition according to the present invention may be combined with a pharmaceutically acceptable carrier as described above.
  • the pharmaceutical composition of the present invention may be prepared in various parenteral or oral dosage forms according to known methods, for parenteral administration
  • Representative formulations are preferably injectable formulations, preferably isotonic aqueous solutions or suspensions
  • injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • Each component may be formulated for injection by dissolving in saline or buffer, and, including but not limited to, oral dosage forms. , Granules, tablets, pills and capsules.
  • compositions formulated in such a manner may be administered in an effective amount via a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular, which is intended to introduce certain substances into a patient in any appropriate manner.
  • the route of administration of the substance may be administered via any general route as long as it can reach the target tissue.
  • the effective amount in the above means an amount exhibiting a prophylactic or therapeutic effect when administered to a patient.
  • the dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient, age, sex, weight, sensitivity to the drug, type of current treatment, administration method, target cells, and the like. It can be easily determined by experts in the field.
  • the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with the conventional therapeutic agent, and may be administered singly or multiplely.
  • all of the above factors can be administered in an amount that can achieve the maximum effect in a minimum amount without side effects, more preferably riOOOO ⁇ g / weight kg / day, even more preferably 10 ⁇ 1000mg / weight
  • the effective dose of kg / day may be repeated several times a day.
  • HDAC6 protein levels in liver cancer samples immunohistochemical staining was performed using monoclonal antibodies against HDAC6 (1: 100, Santa Cruz, Delaware, CA) on tissue microarray samples. Prior to immunostaining, paraffin was removed from the tissue microarray (TMA) slides and hydrated using a series of staged ethanol up to DI water. Endogenous peroxidase activity was blocked by incubating 3% hydrogen peroxide methane in the buffer for 5 minutes. Antigens were diluted by steaming the slides for 20 minutes in a steamer containing boiling sodium citrate buffer (pH 6.0).
  • Human liver cancer cell lines HepG2, Hep3B, PLC PRF / 5, SNU ⁇ 182, SNU-387, SNU-423, and SNU-449 were purchased from American Type Culture Collection (ATCC; Manassas, VA), and human liver cancer cell lines SNU—354. And SNU-368ol from Korean Cell Line Bank (KCLB, Korea).
  • the cells were stored in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (Sigma, St Louis, MO) and 1 mg / ml penicillin / straptomycin (Invitrogen, Grand Island, NY).
  • N-acetyl-D-sphingosine (ceramide), 3-methyladenine (3-MA), and SP600125 were purchased from Sigma.
  • HDAC6 overexpression was confirmed by Western blot analysis.
  • the cells were placed in 6 mm 3 well plates at a concentration of 1> ⁇ 10 5 cells / well in RPMI 1640 medium containing 10% FBS and left for 16-18 hours. After 4 hours of transfection, the medium was replaced with fresh RPMI 1640 medium containing replaced 10% FBS. Cells were then incubated with 5 mg / ml MTT- [3 ⁇ (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliu ⁇ bromide] solution (Sigma) for 1 hour.
  • Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, Calif.) was used to quantify apoptosis levels. Briefly, after trip Xin (tr psinized) the cells, washed twice with cold PBS, IX binding buffer (binding buffer) was suspended (resuspension) reproduction in a concentration of ⁇ ⁇ ⁇ 6 cells / m ⁇ as. Then the above cell suspension containing the 100 ⁇ 5 ⁇ culture ryubeu ( ⁇ ⁇ ⁇ 5 cells to include a) was added to 5 / annexin (annexin) V-FITC and 10 PI (propidium iodide) solution. After 15 minutes of incubation at room temperature in the dark, 400 / ⁇ IX binding buffer (binding buffer) was added to each tube, and apoptosis was measured using a FACSCalibur flow cytometer (BD Biosciences).
  • HDAC6 and Beclin 1 ON-TARGET plus SMART pool human siRNA (HDAC6 and Beclin 1) and Silencer Negative Control siRNA (scrambled siRNA) were purchased from Dharmacon (Lafayette, CO) and Ambion (Austin, TX), respectively.
  • Stable HDAC6 overexpressing Hep3B cells were obtained using trypsin EDTA, plated 1.5> 10 5 cells in a 60 mm dish and grown overnight in incubator. 16-18 hours after plate culture, cells were treated with 100 nmol / L and / or 200 nmol / L HDAC6 and Beclin 1-specific siRNA Transfection was performed with a finite Opti-MEM (Invitrogen). Introduction was performed using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Four to six hours after transduction, the medium was replaced with fresh RPMI 1640 medium containing 10% FBS (Lonza, Basel, Switzerland).
  • Cells were plated with RPMI / 10% FBS on poly-L-lysine coated glass coverslips and then transduced with empty mock HDAC6 expression vector or subject vector. Cells were then washed with PBS, fixed with 4% paraformaldehyde solution in silver for 10 minutes, washed with PBS and permeabilized with 0.2% Triton X-100 for 15 minutes. After blocking the nonspecific binding site with 4% bovine serum albumin in PBS, the cells were washed three times, incubated with LC3B antibody (1: 100) at 4 ° C, and Alexa Fluor 488-linked secondary antibody ( 1: 1000, Invitrogen) was treated for 1 hour at room temperature.
  • LC3B antibody 1: 100
  • Alexa Fluor 488-linked secondary antibody 1: 1000, Invitrogen
  • Hep3B cells stably overexpressing Hep3B cells were cultured in plastic flasks as monolayers, and fixed in 0.1M PBS with 4% paraformaldehyde and 2.5% glutaraldehyde overnight. Cells are then subjected to 1% osmium tetroxide post-fixation, dehydrated with 5 to 100% alcohol series, stained with 0.5% uranyl acetate, and Poly / Bed 812 resin (Pelco, Redding, CA). ), And then thickened. Samples were cut using Ultracut S (Leica, Wetzetlar, Germany) and ultrathin sections were stained with 1% uranyl acetate and 0.2% lead citrate. Cells were observed and photographed with a transmission electron microscope (JEM-1010, Tokyo). ⁇ 1-12> Tumor Xenograft Assay
  • xenograft tumorigenesis assays For xenograft tumors tested (xenograft tumorigenesis assays), it was mixed with ⁇ ⁇ ⁇ (phosphate buffer saline) for 7 cells coming 2 PBS (PH 7.4) and matrigel matrix (BD Biosciences) at the aforementioned target cell line. Cell suspensions were then subcutaneously injected into 4 week old male nude mice. Group 1 was injected with Hep3B cells incorporating a mock, and Groups 2 and 3 were injected with cells introducing Hep3B clones 1 and 2 stably overexpressing HDAC6. Tumor formation at the injection site of mice was examined twice a week. Tumor volume was calculated using the formula 0.5 X length (L) X width 2 (W 2 ). Each experimental group consisted of 5 mice, and tumor growth was quantified by measuring the size of the tumor in three orthogonal directions using calipers. Results are expressed as mean of tumor volume and have a 95% confidence interval.
  • Hep3B cells were plated in 60-mm dishes at a concentration of 5 ⁇ 5 each, allowed to attach overnight, and then transduced. The cells were then treated with PBS containing 10 uM of 5- (and-6) -carboxy-2 ⁇ 7 '-dichlorodihydrofluorescein diacetate (carboxy— H2DCFDA; Invitrogen, Carls bad, CA) for 30 minutes, and CellQuestPro software Flow cytometry was performed with a fitted FACSCalibur (BD Biosciences, San Diego, Calif.).
  • HDAC6 Effect of HDAC6 Expression on Liver Cancer Growth
  • Overexpression of HDAC6 has been reported in many types of cancer cell lines and is known to be required to maintain the transformed traits of many oncogenic cell lines (Aldana-Masangkay GI, Sakamoto KM.The role of HDAC6 in cancer J Biomed Biotechnol; 2011: 875824).
  • the present inventors previously analyzed the HDAC6 expression by microarray analysis based on large-scale transcript change during the development of liver cancer (Nam SW, Park JY, Ramasamy A, Shevade S, Islam A, Long PM, et al.
  • HDAC6 was significantly down-regulated at the site of liver cancer compared to lesions before tumor expression ( Figure 1A). ). Therefore, in order to investigate abnormal expression of HDAC6, the present inventors performed an immunooblot analysis of HDAC6 in human liver cancer tissues. As shown in FIG. 1B, HDAC6 was significantly downregulated in all selected liver cancers as compared to the control non-hepatic cancer tissues. In line with this decrease in HDAC6 expression, the accumulation of alpha-tubulin in a highly acetylated form was observed in liver cancer.
  • HDAC6 expression is downregulated in liver cancer
  • the present inventors measured HDAC6 expression by immunohistochemistry using HCC TMA. Immunostaining results are shown in Table 1, and representative immunostaining results are shown in FIG. 1C.
  • HDAC6 was mostly located in the cytoplasm. Of the 32 normal hepatocellular samples tested, 30 (93.7%) had moderate or strong immunopositive responses to HDAC6 expression, while 15 (46.9%) of 32 liver cancer cases tested Weak stained or negative. In addition, liver cancer (HCC) was never strongly stained for HDAC6. Also found in nine different hepatocellular carcinoma cell lines first established in hepatoblastoma or hepatocellular carcinoma (HepG2, Hep3B, PLC / PRF / 5, SNU182, SNU354, SNU368, SNU387, SNU423, and SNU449). Endogenous expression of HDAC6 was measured by Northern and Western blot analysis (FIG. ID). There were some exceptions, but as expected, human liver cancer cell lines showed relatively low HDAC6 expression (FIG. 1D). These results show that the regulation of HDAC6 was deregulated during cancer development.
  • HDAC6 full-length human HDAC6 cDNA
  • pcDNA_HDAC6 full-length human HDAC6 cDNA
  • HDAC6 did not affect the expression of cell cycle proteins such as pl5INK4B, P 21WAF1 / Cipl, or cyclin-dependent kinase 2 (FIG. 2F). These results indicate that HDAC6 overexpression induces mitotic defects that can be mediated by caspase-independent cell death.
  • HDAC6 a ubiquitin-binding deacetylase
  • HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy.EMBO J; 29: 969—980), we believe that the ethical expression of HDAC6 induces cell death by autophagy in liver cancer. I found out.
  • LC3-II phosphatidylethanolamine
  • Atg8 microtubule-associated protein 1 light chain 3
  • HDAC6 histone deacetylase 6
  • SNU182, SNU368, and SNU449 cell lines with little or no expression of HDAC6 from Northern and Western blot analysis (FIG. 1D).
  • Each cell line was transfected with pcDNA_HDAC6.
  • the growth rate of HDAC6 and expressing cells was lower than the control (non- or mock introducing cells).
  • all cell lines overexpressing HDAC6 showed increased LC3B-II conversion, which was a tumor suppressor due to caspase-independent autophagy cell death of HDAC6. It suggests the function as.
  • Hep3B_HDAC6 clones # 1 and # 2 that stably overexpress HDAC6. Reduction of acetylated alphavtubulin in the cells was detected to confirm overexpression of active HDAC6 (FIG. 5A, left). In addition, gastric cells showed lower growth than mock cells (Hep3B ⁇ Mock) (FIG. 5A, right). In addition, the immunofluorescence assay showed that LC3B accumulation was observed in HDAC6 overexpressing cells (Hep3BAC HDAC6), and LC3B accumulation was hardly observed in control cells (Hep3B ⁇ Mock) (FIG. 5B).
  • Beclin 1 was involved in the early stages of autophagy, promoting the crystallization of autophagy vesicles and the recruitment of proteins from the cytoplasm.
  • Beclin 1 was involved in the early stages of autophagy, promoting the crystallization of autophagy vesicles and the recruitment of proteins from the cytoplasm.
  • Beclin 1 expression is directly related to autophagosome production and that Beclin 1 interacts with antiapoptotic Bel-2 family members Bcl-2 and Bcl-xL ( Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature 1999; 402: 672—676).
  • the present invention shows that HDAC6 overexpression in Hep3B cells mediates cell death by autophagy via the activity of the Beclin 1 and LC3 ′ II pathways.
  • HDAC6 overexpressing cells expressed much more Beclin 1 and LC3B-II than Hep3B_Mock cells. This effect of HDAC6 on autophagy was confirmed by treating cells with ceramide, which potentially induces apoptosis by gastric cell lines.
  • HDAC6 was inactivated with siRNA directed to it. As expected, HDAC6 knockdown significantly inhibited Beclin 1 expression and conversion of LC3B—I to LC3B-II (FIG. 6B).
  • 3—MA On HDAC6 overexpressing cells.
  • Hep3B_HDAC6 cells treated with 3-MA inhibited Beclin 1 and LC3B-II expression.
  • Beclin 1 knockdown on HDAC6-mediated autophagy we examined the effect of Beclin 1 knockdown on HDAC6-mediated autophagy.
  • Beclin 1 knockdown in HDAC6 overexpressing cells (Hep3B_HDAC6 clones # 1 and # 2) significantly inhibited LC3B-II production.
  • HDAC6 shows tumor suppression effects through Beclin 1 and LC3—II processing dependent autophagy in liver cancer cells.

Abstract

La présente invention concerne l'utilisation de HDAC6 (histone désacétylase 6) en tant que marqueur de diagnostic de l'hépatome. Plus spécifiquement, la présente invention concerne un marqueur de diagnostic de l'hépatome à l'aide d'un gène ou une protéine HDAC6, un kit de diagnostic de l'hépatome l'utilisant, et un procédé de diagnostic de l'hépatome l'utilisant, et se rapporte également à un procédé de criblage pour une substance capable de prévenir ou guérir l'hépatome par la récupération de l'expression de gène HDAC6, et à une composition de prévention ou de traitement de l'hépatome comprenant la substance. Le gène HDAC6 selon la présente invention a été confirmé de façon à réduire la quantité de l'expression dans des tissus ou des cellules d'hépatome par rapport à des tissus ou des cellules non-hépatomes. Par conséquent, l'utilisation du gène HDAC6 en tant que marqueur de diagnostic de l'hépatome rend possible le diagnostic de façon rapide et correcte de l'hépatome à un stade précoce, et, en outre, l'invention peut être utilisée en tant qu'agent thérapeutique pour l' hépatome.
PCT/KR2012/011549 2012-02-14 2012-12-27 Utilisation de hdac6 en tant que marqueur d'hépatome et agent thérapeutique WO2013122321A1 (fr)

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