Study On The Role And Molecular Mechanism Of CDK9 In Hepatocellular Carcinoma Metastasis

Background Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide with an increasing mortality.Although considerable progress has been made in the prevention,surveillance,early detection,diagnosis and treatment,the long-term prognosis of HCC is still not satisfactory because of high rates of recurrence and distant metastasis.Therefore,exploring the underlying mechanisms and novel therapeutic targets hold promise for the reduction of HCC burden.Tumor-promoting inflammation and immune escape are hallmarks of cancer.HCC develops within a pre-existing liver chronic inflammation and is a typical inflammationrelated cancer with infiltration of immune cells.Immune escape is often accompanied by the recruitment of immunosuppressive cells such as myeloid-derived suppressor cells(MDSCs)and tumor-associated macrophages(TAMs).It is well established that MDSCs and TAMs exert pro-tumorigenic effects by suppressing T cell function and promoting tumor angiogenesis,proliferation and metastasis.However,how these cells are recruited in HCC remains largely unclear.Cyclin-dependent kinases(CDKs),a family of serine/threonine kinases,are responsible for series of critical cellular processes including proliferation,differentiation,DNA repair and apoptosis through regulating the cell cycle and gene transcription,when a catalytic kinase subunit binds to a regulatory cyclin subunit to form the heterodimeric complexes.Recent studies have deciphered that frequent dysregulation of CDK family genes is associated with tumorigenesis and progression of different cancer types,which makes them attractive therapeutic targets.CDK9 has been demonstrated to play essential roles in several physiological and pathological pathways through binding to cyclin T and forming P-TEFb,which is able to phosphorylate the C-terminal domain(CTD)of RNA polymerase II on serine-2 to regulate transcription elongation.CDK9 has been confirmed to promote tumor cell proliferation,migration and invasion,which makes CDK9 such an important target.However,the mechanism underlying the oncogenic function which CDK9 played in HCC progression and metastasis needs further elucidation.Objectives: 1.To examine the expression levels of CDK family genes in HCC.2.To examine the expression and function of CDK9 in HCC.3.To investigate the expression of MDSCs and TAMs in HCC.4.To study the underlying mechanism of CDK9 in promoting HCC metastasis.5.To study the potential therapeutic strategy to inhibit HCC metastasis.Methods: 1.The expression levels of CDK family genes in HCC and adjacent nontumor tissues were detected by RT-PCR.2.The expression of CDK9 in HCC and adjacent nontumor tissues was detected by RTPCR and IHC.3.Cell lines of CDK9 overexpression and knockdown were established by lentiviral transduction.The function of CDK9-mediated invasion and metastasis was analyzed using Transwell assays and liver orthotopic metastasis model.4.The Chemokines and Receptors PCR Array was utilized to screen the possible downstream targets of CDK9.The changes of target genes were verified by Western blotting.The effect of targeted gene in CDK9-induced HCC invasion and metastasis was verified using liver orthotopic metastasis model.5.The infiltration of MDSCs and TAMs was investigated using liver orthotopic metastasis model and immunofluorescence.6.The effects of administration of CDK9 inhibitor and/or CCR2 inhibitor were detected using liver orthotopic metastasis model and immunofluorescence.Results: 1.The expression levels of CDK1,CDK4,CDK5,CDK6,CDK7,CDK9,CDK12,CDK14,and CDK20 were significantly increased in HCC tissues than those adjacent nontumor tissues.Among these upregulated CDKs,CDK9 was identified as the relatively largest foldchange gene.Therefore,we further investigate the pivotal role of CDK9 in HCC.2.The m RNA and protein expression of CDK9 was significantly higher in HCC tissues than in adjacent nontumor tissues.HCC tissues from patients with recurrence or metastasis exhibited obviously increased m RNA expression of CDK9 than those patients without recurrence or metastasis.Further,we established two stable cell lines,Hep3B-CDK9 and MHCC97H-sh CDK9 with lentiviral infection.Transwell assays showed that elevated CDK9 expression increased the migratory and invasive abilities of Hep3 B cells,while knockdown of CDK9 resulted in the opposite effects.In vivo metastasis assay showed that CDK9 overexpression facilitated lung metastasis of Hep3 B cells,while CDK9 knockdown suppressed the lung metastasis of MHCC97 H cells.3.The Chemokines and Receptors PCR Array revealed that CDK9 upregulated the expression of CCL2(MCP-1).Furthermore,we established two stable cell lines,Hepa1-6-CDK9 and Hepa1-6-CDK9+LV-sh MCP-1 with lentiviral infection.In vivo metastasis assay showed that CDK9 overexpression facilitated the lung metastasis of Hepa1-6 cells,while downregulation of MCP-1 repressed the CDK9-induced lung metastasis.4.The migratory abilities of MDSCs and TAMs were significantly increased after treating with conditioned medium from Hepa1-6-CDK9 cells than treating with conditioned medium from Hepa1-6-control cells.Either MCP-1 knockdown or treatment of CCR2 inhibitor RS102895 significantly impaired the migratory ability of MDSCs and TAMs induced by CDK9 overexpression.Further,CCR2 inhibitor RS102895 reduced the lung metastasis induced by CDK9 overexpression,and immunofluorescence showed that CCR2 inhibitor RS102895 suppressed the infiltration of MDSCs and TAMs,and improved the amount of CD8+T cells.Next,we performed the anti-Gr-1 antibody and clodronate liposomes to deplete the MDSCs and TAMs,and found that the infiltrating of MDSCs and TAMs was suppressed and the amount of CD8+T cells was increased.And immunofluorescence also showed the similar results.In addition,the expression of CDK9 is positively correlated with CCL2 and intratumoral MDSCs and TAMs infiltration in human HCC tissues.5.Combined treatment of CDK9 inhibitor MC180295 and CCR2 inhibitor RS102895 dramatically decreased CDK9-driven HCC metastasis in vivo.Immunofluorescence showed that combined treatment reduced the amount of MDSCs and TAMs,and improved the amount of CD8+T cells.Conclusion: In the present study,we investigate the expression of CDK9 in HCC and the role of CDK9 in HCC metastasis.CDK9 promoted the accumulation of MDSCs and TAMs and facilitated HCC metastasis by CCL2/CCR2 pathway.Combined treatment of CDK9 inhibitor MC180295 and CCR2 inhibitor RS102895 dramatically decreased CDK9-driven HCC metastasis.Thus,CDK9 is a prognostic biomarker in human HCC.