SIRT1 Facilitates Hepatocellular Carcinoma Metastasis By PromotingPGC-1α-mediated Mitochondrial Biogenesis

BackgroundHepatocellular carcinoma(HCC) is the fifth most common cancer malignancy and the third leading cause of cancer-related mortality. The majority of the HCC patient was diagnosed in advanced stages and appeared intrahepatic and extrahepatic metastases. Although several advanced therapeutic approaches have been applied for the treatment of HCC, such as surgical resection, radio-frequency, microwave ablation, chemotherapy and liver transplantation, due to the metastasis in early stage and frequent recurrence after surgery, the prognosis of HCC is extremely poor, the survival rate of 5-year is only 40%-50%. Therefore, metastasis and recurrence have been the key factors affecting prognosis of HCC, exploring novel metastatic mechanism and disrupting it are great significant to improve the prognosis of HCC patients. The emerging reports indicated that SIRT1 has the Janus-faced role in tumorigenesis. Overexpression of SIRT1 promoted the invasion and metastasis of prostate cancer cells, pancreatic cancer cells and melanoma cells, but SIRT1 up-regulation inhibited the migration and invasion of oral squamous cell carcinoma cells. However, how SIRT1 affect the metastasis and prognosis of HCC is still ambiguous. After determining the SIRT1 expression patterns and the correlation between SIRT1 level and clinical prognosis, we discovered that SIRT1 influence the prognosis of HCC through facilitating the invasion and metastasis. After demonstrating the SIRT1 physically interact with PGC-1α, we clarified the novel mechanism that SIRT1 facilitated HCC metastasis by promoting PGC-1α-mediated mitochondrial biogenesis.Methods(1)The liver tumor tissues and corresponding adjacent nontumoral tissues were collected from 72 HCC patients with different stages during curative hepatectomy at The Xin Qiao Hospital of Third Military Medical University, simultaneously, the normal liver tissues were collected from 5 patients without liver disease. The partial tissues were stored at-80°C immediately after resection for extracting protein and RNA, the other tissues were fixed by 4% paraformaldehyde, and the paraffin-embedded samples were freshly cut into 5-mm sections before immunohistochemistry. Western blot analysis detected the protein level of SIRT1 in liver tumor tissues and various HCC cell lines, Real Time PCR assay detected the mRNA level of SIRT1 in HCC specimens, immunohistochemistry analyzed the location and intensity of SIRT1 expression in liver tumor tissues, corresponding adjacent nontumoral tissues and normal liver tissues.(2)Lentivirus transfection and subsequently screening with puromycin were adopted to build stable transfected HCC cell lines. CCK-8 kit was used to detect the cell viability, Cell-LightTM EdU Apollo 567 in vitro kit was apply to determine the proliferation of cells, the subcutaneous transplantation assay detected that how SIRT1 affect the tumorigenesis of HCC cells. Wound healing assay and Transwell migration assay were used to detect the effect of SIRT1 on the migratory ability of HCC cell lines, and the effect of SIRT1 on the invasive ability of HCC cell lines was investigated by Transwell invasion assay. The tail vein injection assay was applied to assess the effect of SIRT1 knockdown on the metastatic ability of HCC cells in vivo. The Bioluminescence Imaging and Analysis was used to monitor the effect of SIRT1 silencing on the metastatic foci formation in whole body. The hematoxylin and eosin staining detected the tumor nodules in lung tissues derived from HCC cells.(3)Immunofluorescence staining detected the expression of EMT markers CK-18, vimentin, fibronectin in HCC cells. NAO staining and MitoTracker? Red CM-H2 Xros probes staining were used to evaluate the effect of SIRT1 knockdown on the mitochondrial mass. The E.Z.N.ATM DNA kit was applied to extract the total DNA from cells and tissues of nude mice, then Real Time PCR analyzed the mtDNA copy number and the mRNA level of mtDNA encoding genes(COX I, ND1, ND6). The ATP assay kit detected the cellular ATP levels in sh-SIRT1-MHCC97 H cells and overexpression of PGC-1α in sh-SIRT1-MHCC97 H cells. Co-immunoprecipitation assay determined that SIRT1 could deacetylate PGC-1α and affect its activity. The liver orthotopic implantation model was used to assessed that the effect of SIRT1 depletion and overexpression of PGC-1α on the pulmonary metastasis of HCC cells. The Bioluminescence Imaging and Analysis was used to monitor the location and times of the metastatic foci formation from the HCC cells in nude mice.(4)Statistical analysis. Quantitative comparisons of the relative protein and mRNA level of SIRT1 between the HCC tissue and adjacent nontumoral liver tissues were analyzed by the paired Student’s t test. The correlations between relative expression level of SIRT1 and the clinicopathological parameters were evaluated using the nonparametric chi-squared test and Spearman’s rank test according to different group. The disease-free survivals and overall survival rates were calculated from the date of tumor resection to the time of first recurrence(disease-free survival) or death(overall survival), and the Kaplan-Meier’s analysis was used to estimate the disease-free survivals and overall survival rates according to SIRT1 expression level. The data of two groups were analyzed by Student’s t test, and the data between the several groups were compared using one-way ANOVAs. All of the experimental data are expressed as the means ± SEM, and the statistical analyses were performed using SPSS software(version 13.0). A two-tailed P value<0.05 was regarded as statistically significant.Results(1)Elevated SIRT1 expression was frequently observed in the HCC cell lines compared to the L02 cells, an immortalized human liver cell line. The mRNA levels of the SIRT1 were significantly increased in the HCC tissues compared to the adjacent nontumoral liver tissue(n = 24, P = 0.005). SIRT1 protein level was frequently up-regulated in HCC tissues, Overexpression of SIRT1(defines as a > 2-fold increase) was detected in 56.9%(41/72) of HCC tumors compared to the corresponding nontumoral tissues. the levels of the SIRT1 protein were apparently up-regulated in the HCC tissues compared to the paired adjacent nontumoral liver(n = 72,P = 0.012). The immunohistochemical(IHC) analyses showed that SIRT1 was highly expressed in the HCC tumors compared to the adjacent nontumoral tissues and normal liver tissues, and was primarily localized in the nucleus. The correlation analysis between SIRT1 expression and clinical parameters indicated that the increased SIRT1 expression was significantly associated with portal vein tumor thrombus(P = 0.0039) and the HCC patients with advanced tumor stages(P = 0.0016), but the relative expression of SIRT1 was not correlated with the other clinicopathological features, such as age, sex, HBV infection, Child-Pugh class,Cirrhosis, serum AFP level, tumor size, Edmondson-Steiner grade, Milan criteria. Kaplan-Meier’s analysis showed that HCC patients with overexpression of SIRT1 had shorter DFS(disease free survival, P = 0.021) and worse OS(overall survival, P = 0.039) than those patients without SIRT1 overexpression. These results indicated that SIRT1 expression level could serve as a valuable index for predicting recurrence and poor survival of HCC patients.(2)Overexpression of SIRT1 or depletion of SIRT1 in HCC cells did not affect the cell viability and proliferation. SIRT1 shRNA-expressing MHCC97 H tumors showed similar tumor growth kinetics, weights and volume compared to the control shRNA-expressing MHCC97 H tumors. These results indicated that SIRT1 expression level does not have an effect on HCC proliferation and tumorigenesis. The wound-healing ability of the stable SIRT1 knockdown MHCC97 H cells was significantly attenuated compared to the shRNA vector-transfected cells(P < 0.01). SIRT1 knockdown markedly decreased the migratory capacity(P < 0.01) and impaired cells invasion of the MHCC97 H through the Transwell chamber assay. In addition, SIRT1 overexpression significantly enhanced the migration and invasion capacities of liver cell line L02(P < 0.05). Taken together, these results suggested that SIRT1 expression increased the migratory and invasive capacities of HCC cells in vitro. In the tail vein injection assay, the fluorescence signaling analysis showed that SIRT1 knockdown led to a dramatic reduction in distant organ metastasis compared to the shRNA control cells(P < 0.01). A significantly smaller number of metastatic nodules were induced on the surface of the livers from the mice injected with the MHCC97H-sh-SIRT1 cells than on those with the MHCC97H-sh-control cells(P < 0.01). The incidence of lung metastasis in the MHCC97H-sh-SIRT1 group was significantly decreased compared to that in the control group cells. Taken together, SIRT1 expression promoted HCC invasion and metastasis.(3)After down-regulating the SIRT1 levels in the MHCC97 H and SK-Hep1 cells and up-regulating the SIRT1 levels in the Huh7 cells, the western blot assay indicated that there were no significant variations in the epithelial markers(E-cadherin and CK-18), mesenchymal markers(vimentin and α-SMA) and transcriptional factors(twist and snail). There were no significant changes in the mRNA levels of the key EMT markers(E-cadherin, fibronectin, vimentin, twist, and snail) in the SIRT1-depleted MHCC97 H cells compared to the sh-control-MHCC97 H cells. The immunofluorescence staining indicated that SIRT1 silencing did not induce the EMT program in the MHCC97 H and SK-Hep1 cells. SIRT1 did not induce the EMT in HepG2 cells transfected with LV-SIRT1 in hypoxic conditions. In the EMT model of HCC cells induced by TGF-β1, the SIRT1 levels were not changed. All of these results suggested that SIRT1 expression did not induce the EMT program in HCC cells. After knockdown of SIRT1 in MHCC97 H cells stably, the mitochondrial mass, mtDNA copy number, mtDNA transcripts(COX I, ND1, ND6), cellular ATP levels and the protein level of mitochondrial biogenesis related encoding genes(Tfam, COX IV, TOMM20) were significantly reduced. These results revealed that SIRT1 depletion in HCC cells reduced the capacity for mitochondrial biogenesis and impaired mitochondrial function, leading to deficient bioenergetic production. Western blot assay analyzed the HCC specimens and showed that SIRT1 overexpression was highly associated with the up-regulation of PGC-1α in the HCC tumors compared to the adjacent nontumoral liver(r = 0.569,P < 0.001). Overexpression of SIRT1 in the Huh7 and MHCC97 H cells significantly increased the PGC-1α expression level, and depletion of SIRT1 in MHCC97 H cells significantly increased the acetylated-PGC-1α level, and reduced the activity of PGC-1α. The ectopic expression of PGC-1α in the Huh7 and MHCC97 H cells significantly increased the cells’ migration and invasion, enhanced the mtDNA copy number and the mRNA level of mtDNA encoding genes(COX I,ND1,ND6), these results demonstrated that t PGC-1α promoted HCC cells migration and invasion by enhancing mitochondrial biogenesis. Ectopic expression of PGC-1α significantly restored cell migration and enhanced the cellular invasion that was inhibited by SIRT1 depletion. The levels of mtDNA copy number, mt DNA transcripts(COX I,ND1,ND6), cellular ATP and protein of mitochondrial biogenesis related genes(Tfam,COX IV,TOMM20) were significantly increased after PGC-1α was overexpressed in the SIRT1-depleted MHCC97 H cells compared to those in SIRT1-depleted MHCC97 H cells. These results suggested that PGC-1α overexpression reversed the inhibitory effects of SIRT1 knockdown on invasion and mitochondrial biogenesis. In the orthotopic transplantation model of nude mice, the intensity of fluorescence photo flux and the number of tumor nodules in lung tissues were significantly reduced in SIRT1-depleted MHCC97 H cells compared to the control cells, but this inhibitory effect could be reversed by overexpressing PGC-1α. The levels of mtDNA copy number, mtDNA transcripts(COX I,ND1,ND6) and mitochondrial biogenesis related genes(Tfam,COX IV,TOMM20) proteins derived form the metastatic lung tissues of nude mice were significantly reduced in the MHCC97H-sh-SIRT1 group compared to those in MHCC97H-sh-control group, but PGC-1α overexpression restored these levels. These results suggested that the PGC-1α-mediated increase in mitochondrial biogenesis plays a critical role in promoting SIRT1 overexpression-facilitated HCC metastasis.Taken together, SIRT1 overexpression was frequently detected in HCC tissues and the relative level of SIRT1 was significantly associated with microvascular invasion, TNM stages, HCC recurrence and overall survival rate, which suggested that SIRT1 expression levels could be an effective prognostic biomarker for evaluating metastasis in HCC. Knockdown of SIRT1 in HCC cells produced a significant reduction in invasion and metastasis in vitro and in vivo. The further study of mechanism revealed that SIRT1 depletion attenuated mitochondrial biogenesis and caused deficiency of ATP production but did not affect the status of the epithelial-mesenchymal transition program. Elevated SIRT1 expression was highly correlated with an up-regulation of PGC-1α in the HCC specimens. Ectopic expression of SIRT1 in HCC cells increased the PGC-1α levels, and knockdown of SIRT1 reduced the activity of PGC-1α. The cell assays and orthotopic transplantation model showed that PGC-1α overexpression reversed the inhibitory effects of SIRT1 depletion on invasion and metastasis by enhancing mitochondrial biogenesis. Taken together, these findings clarified a novel mechanism of SIRT1-facilitated HCC metastasis and provide a rationale to explore a highly efficient therapeutic target against the SIRT1/PGC-1α axis, which enriched the theory of individualized treatment of HCC and has positive significance for improving prognosis.