Interleukin 1β-mediated HOXC10 Overexpression Promotes Hepatocellular Carcinoma Metastasis By Upregulating PDPK1 And VASP

Background:Hepatocellular carcinoma(HCC),a highly aggressive primary liver cancer,ranks as the fifth most common malignancy worldwide and the second leading cause of cancerrelated death.Surgical resection is still the best therapeutic strategy for patients at early stage,but many HCC patients develop postsurgical recurrence or metastasis with poor 5-year survival rates.The high rates of tumor recurrence and distant metastasis after surgical resection are the main reason for the poor prognosis of patients with HCC.Therefore,it is still eagerly need to explore the molecular mechanism underlying HCC metastasis.Homeobox(HOX)genes are a highly conserved subgroup of the homeobox superfamily that plays an important role in embryonic development and physiological processes,such as apoptosis,differentiation,receptor signaling,motility and angiogenesis.In mammals,39 HOX genes have been identified and classified in four separate clusters(A,B,C and D).In recent years,mounting evidence has indicated that the deregulation of HOX family genes plays critical roles in cancer initiation and progression.For example,overexpression of HOXB7 and HOXA13 promotes cancer invasion and metastasis and indicates poor prognosis.In contrast,HOXA5 and HOXA9 are downregulated in human cancer and function as tumor suppressor genes(TSG).These studies indicate that HOX family genes are master regulators of cancer progression and metastasis.However,the function of HOXC10 gene in HCC remains unknown.In mammals,HOXC10 regulates the cell cycle,mitotic progression,and embryonic development.Deregulated HOXC10 functions as an oncogene and contributes to malignant behaviors in several human cancers,including glioma,cervical squamous cell carcinoma and gastric cancer.However,whether HOXC10 is involved in HCC progression and metastasis remain unknown.Objectives:1.To detect the expression and function of HOX family in HCC.2.To investigate the expression pattern of HOXC10 in HCC.3.To investigate the function of HOXC10 in HCC.4.To study the underlying mechanism of HOXC10 promoting HCC metastasis.5.To investigate the mechanism of HOXC10 upregulation in HCC.6.To study the potential therapeutic strategy to inhibit HCC metastasis.Methods:1.The expression of HOX family in tissues of normal liver,adjacent nontumor and HCC was detected by RT-PCR.The function of the elevated HOX genes-mediated invasion and metastasis was analyzed by the Transwell assays.2.The expression of HOXC10 in tissues of normal liver,adjacent nontumor and HCC was detected by RT-PCR and immunohistochemical staining.3.The HOXC10 overexpression and knockdown cell lines were established by lentiviral transduction.The effect of HOXC10-mediated invasion and metastasis was analyzed using Transwell assays and liver orthotopic metastasis model.4.The Affymetrix Prime View Human Gene Expression Array was utilized to screen possible HOXC10 downstream targets.The change of target genes upon HOXC10 upor down-regulation was verified by western blot and RT-PCR.Luciferase reporter and chromatin immunoprecipitation(Ch IP)assays were performed to measure the transcriptional and binding of HOXC10 to the promoters of PDPK1 or VASP.5.We treated Hep3 B and PLC/PRF/5 cells with different inflammatory factors.Luciferase reporter assay was used to investigate the role of cis-regulatory elements of the HOXC10 promoter in response to IL-1β stimulation.Western blot assays were utilized to investigate the change in HOXC10 expression in response to IL-1β treatment and ERK(SCH772984),P38 kinase(SB202190)and JNK(SP600125)signaling inhibition.The Ch IP assay was performed to further determine transcription of c-Jun to the promoter of HOXC106.We downregulated HOXC10 expression via lentiviral transduction in Hep3B-IL-1β cell,the effect of HOXC10 in IL-1β-mediated invasion and metastasis were analyzed by in vitro Transwell assays and in vivo liver orthotopic metastasis model.We determine the Anakinra treatment effects on IL-1β-HOXC10 signaling-mediated HCC invasion and metastasis by in intro and in vivo experiments.Results:1.The expression levels of HOXA3,HOXA6,HOXA10,HOXA13,HOXB5,HOXB7,HOXC4,HOXC10 and HOXD9 were significantly higher in HCC tissues than in adjacent nontumor tissues.Transwell assays indicated that cell migration and invasion were remarkably inhibited in HCCLM3 cells by the downregulation of HOXC10.Therefore,we focused on the HOXC10 gene for further study.2.The m RNA level of HOXC10 was dramatically higher in HCC tissues than in adjacent nontumor tissues and normal liver tissues.Notably,HOXC10 expression was significantly higher in patients with recurrence than in patients without recurrence.Furthermore,the HOXC10 expression level was much higher in patients with metastasis than in patients without metastasis.We establish two stable cell lines,Hep3B-HOXC10 and HCCLM3-sh HOXC10 through lentivirus transduction.Transwell assays showed that HOXC10 overexpression increased the migration and invasion abilities of Hep3 B cells,while knockdown of HOXC10 resulted in the opposite effects.In vivo metastasis assay showed that HOXC10 upregulation increased the HCC cells lung metastasis,while HOXC10 downregulation markedly decreased the HCC cells lung metastasis.3.Affymetrix Prime View Human Gene Expression Array revealed that HOXC10 overexpression resulted in upregulated expression of a series of metastasis-related genes,including PDPK1,VASP,BMP6 and KRT17.Considering the important role of PDPK1 and VASP in cancer invasion and metastasis,we focused on PDPK1 and VASP for further study.Overexpression of HOXC10 significantly upregulated PDPK1 and VASP expression,whereas knockdown of HOXC10 reduced the expression levels of both genes.Serial deletion and site-directed mutagenesis luciferase reporter assay showed the binding site 2/1 and 1 was the binding site of HOXC10 in PDPK1 and VASP promoter,respectively.Furthermore,the Ch IP assay demonstrated that HOXC10 binding was indeed enriched in these regions in both HCC cell lines and human HCC tissues.4.Transwell assays showed that knockdown of PDPK1 and VASP significantly suppressed HOXC10-mediated migration and invasion capacities,while overexpression of PDPK1 and VASP rescued the reduced migration and invasion abilities of HCCLM3 cells with HOXC10 knockdown.An in vivo metastasis assay showed that knockdown of PDPK1 and VASP reduced the incidence of lung metastasis and the number of metastatic nodules of the Hep3B-HOXC10 group.In contrast,upregulation of PDPK1 and VASP reversed the suppression of HCC metastasis in the HCCLM3-sh HOXC10 group.In human HCC tissues,HOXC10 expression was positively correlated with PDPK1 and VASP expression.5.Hep3 B and PLC/PRF/5 cells with low endogenous HOXC10 were treated with recombinant IL-1β,IL-6,IL-8,IL-17 A,TNF-α and TGF-β.IL-1β was the most powerful inducer of HOXC10 expression in both cells.The luciferase report experiment showed that the c-Jun binding site(located at the-359 site)was the regulation region of IL-1βin HOXC10 promoter.Pretreatment of cells with the JNK inhibitor rather by ERK or P38 inhibitors significantly decreased IL-1β-mediated HOXC10 expression.Consistently,a Ch IP assay demonstrated that the JNK inhibitor markedly inhibited the binding of c-Jun to the HOXC10 promoter.These data suggested that IL-1β upregulated HOXC10 expression through the JNK/c-Jun signaling pathway.6.Transwell results revealed knockdown of HOXC10 dramatically decreased cell migration and invasion abilities of Hep3B-IL-1β cells.In vivo metastasis assay,downregulation of HOXC10 decreased the incidence of lung metastasis and the number of metastatic lung nodules while increasing the overall survival in the Hep3B-IL-1βxenograft group.Anakinra,a specific antagonist of IL-1R1,inhibited IL-1β-induced HOXC10 upregulation and HCC metastasis by both in vitro and in vivo assay.In human HCC tissues,HOXC10 expression was positively correlated with IL-1R1 expression.Conclusion:In the present study,we demonstrate that HOXC10 promoted HCC metastasis by upregulating PDPK1 and VASP expression.IL-1β/IL-1R1 signal upregulated HOXC10 expression via the JNK/c-Jun pathway.Anakinra,a specific antagonist for IL-1R1,inhibited IL-1β-induced HOXC10 upregulation and HCC metastasis.Thus,HOXC10 is a prognostic biomarker in human HCC,and targeting this signaling pathway may provide evidence for the development of potential treatment strategies for HCC.