CAMSAP2-mediated Noncentrosomal Microtubule Acetylation Promotes Hepatocellular Carcinoma Metastasis And Indicates Poor Prognosis

BACKGROUND & AIMS: Hepatocellular carcinoma(HCC)is the sixth most common neoplasm and the third leading cause of cancer-related death worldwide.The poor prognosis of HCC patients mainly results from the high frequencies of distant metastasis and tumor recurrence after curative resection.Though diverse microtubule(MT)-targeting agents have been part of the pharmacopoeia of anticancer therapy for decades,they have little effect on HCC metastasis.Therefore,exploring MT nucleation and distribution patterns in HCC cells may provide new insights into the suppression of HCC metastasis.Significant advances have been made in understanding the function and dysfunction of+TIPs.In comparison,studies of-TIPs,such as CAMSAPs and Patronin,have only just begun to emerge.CAMSAP2,a member of the CAMSAP/Nezha/Patronin family,can be specifically deposited on MT minus-ends and serve as “seeds” for noncentrosomal MT outgrowth.Depletion of CAMSAP2 alters the assembly pattern of MTs,resulting in the transformation of the partially noncentrosomal MT network into a completely radial centrosomal array.Further,previous studies have demonstrated that CAMSAP2 is important for cell polarization,intracellular transport,and organelle assembly.Importantly,CAMSAP2 depletion was shown to impair directional cell migration,a result in line with previous observations that suggested that Golgi-anchored noncentrosomal MTs are important for efficient cell movement.These intriguing findings raise the question of whether cancer cells can also promote directional migration by maintaining noncentrosomal MTs and suppressing the MT-organizing ability of the centrosome.METHODS: The expression of CAMSAP2,HDAC6 and acetylated ?-tubulin were analyzed by immunohistochemistry in two independent HCC cohorts.Immunofluorescenceand Co-immunoprecipitation were used for structural,morphological and biochemical analyses of CAMSAP2-mediated noncentrosomal microtubule.In vitro migration and invasion assays and in vivo metastatic colonization assays were utilized to investigate the roles of CAMSAP2 in HCC invasion and metastasis.RESULTS:Part I: The Cancer Genome Atlas(TCGA)datasets revealed that the messenger RNA(mRNA)expression of CAMSAP2 was significantly increased in liver cancer specimens.Further,Kaplan-Meier analysis based on TCGA dataset revealed that liver cancer patients with high levels of CAMSAP2 mRNA had a significant shorter overall survival(OS)and disease-free survival(DFS).We analyzed CAMSAP2 expression by immunohistochemistry in two tissue microarrays of HCC samples.The results showed that CAMSAP2 was dramatically up-regulated in HCC tissues.Up-regulation of CAMSAP2 was confirmed in an additional 90 paired HCC samples by real-time PCR.CAMSAP2 mRNA expression was also significantly increased in HCC tissues.Moreover,Western blotting and IF showed that CAMSAP2 was up-regulated in HCC tissues.Further analysis of the clinicopathological characteristics of paired HCC tissues showed that CAMSAP2 overexpression was highly associated with multiple tumors,increased tumor size,microvascular invasion,poor tumor differentiation,and a higher tumor-nodule-metastasis(TNM)stage.HCC patients with high CAMSAP2 expression had a higher recurrence and a shorter OS.Taken together,these data indicate that CAMSAP2 is a potential prognostic biomarker for the malignant progression and metastasis of HCC.Part II: RT-PCR and Western blotting indicated that CAMSAP2 expression in HCC cell lines with high metastatic potential was substantially higher than that in the hepatocyte line and in HCC cell lines with low metastatic potential.Depletion of CAMSAP2 markedly reduced HCC cell migration and invasion,as evaluated using a wound-healing assay.Conversely,CAMSAP2 up-regulation significantly enhanced the migration and invasion capacities of MHCC97 H and Huh7 cells.Similar results were confirmed using a transwellassay.In three-dimensional(3D)cultures,IF staining indicated that CAMSAP2 promotes HCC cell invasion in an in vivo-like environment.To assess the effect of CAMSAP2 on tumor metastasis in vivo,cells were injected into the livers of nude mice to establish an orthotopic metastatic model.Consistent with our in vitro results,CAMSAP2 up-regulation significantly increased intrahepatic and lung metastasis,resulting in a shorter OS time.In contrast,CAMSAP2 down-regulation reduced the incidence of lung metastasis and the number of metastatic pulmonary nodules,resulting in a longer OS time.Collectively,these lines of evidence suggest that CAMSAP2 significantly promotes HCC metastasis.Part ?: Double immunoflurence staining for CAMSAP2 and ?-tubulin and endogenous co-immunoprecipitation(co-IP)assays demonstrate that CAMSAP2 can form CAMSAP-decorated noncentrosomal MT stretches in HCC cells.Western blotting and real-time PCR showed that down-regulation of CAMSAP2 significantly increased the expression of ?-tubulin.Immunostaining revealed that depletion of CAMSAP2 transformed the partially noncentrosomal MT network into a completely radial centrosomal array wherein the centrosome relocated around the center of nucleus in HCC cells.In CAMSAP2-depleted cells,the Golgi apparatus lost its typical ribbon-like morphology clustered around the centrosome.The MT system became more radial and failed to co-localize with the Golgi stacks.IF staining showed that the initial repolymerization of MTs radiated from the centrosome 15 min after drug washout;however,there were no visible noncentrosomal MTs in CAMSAP2-depleted cells.These observations suggest that CAMSAP2 can cluster at MT minus end and form CAMSAP2-decorated noncentrosomal MTs in HCC cells.As anticipated,loss of CAMSAP2 induced defects in directional cell migration.CAMSAP2 depletion caused cells to fail to polarize properly,as characterized by defective Golgi reorientation towards the wound edge,and affected the formation of protrusions at the leading edge compared with that in control cells.These results suggest that CAMSAP2-dependent MT organization contributes to directional cell migration in HCC.Part ?: Western blotting showed that MT acetylation was markedly reduced in CAMSAP2-knockdown HCC cells.Conversely,CAMSAP2 overexpression elevated the level of acetylated ?-tubulin.Further,IF staining showed that the acetylated MT network,which typically extends protrusions towards the direction of migration,was disrupted in CAMSAP2-knockdown cells.Acetylation of MTs is tightly controlled by multiple acetyltransferases and deacetylases.Western blotting analysis showed that CAMSAP2down-regulation significantly increased the expression of HDAC6 and SIRT2 with no apparent change in the expression of ?-TAT1.Whereas,the expression of HDAC6 and SIRT2 obviously decreased in CAMSAP2 overexpression.Next,we used specific inhibitors against HDAC6(tubacin)and SIRT2(thiomyristoyl,TM)to inactivate HDAC6 and SIRT2,respectively.Western blotting analysis showed that the addition of tubacin but not TM elevated the level of MT acetylation in a dose-dependent manner.RNAi of HDAC6 confirmed that down-regulation of HDAC6 increased the MT acetylation level.Further,immunostaining revealed that inactivation of HDAC6 up-regulated the acetylation level of?-tubulin.Closer observation revealed that,after tubacin or RNAi treatment,acetylated?-tubulin aligned with the MT network and extended towards the leading edge of cell protrusions.Transwell assay showed that inactivation of acetylated MTs by RNAi or pretreatment with tubacin dramatically increased the migration and invasion capabilities of HCC cells.These results suggest that CAMSAP2 promotes HCC cell migration and invasion by elevating the acetylation level of MTs.Part ?: Western blotting analysis also showed that the inhibition of HDAC6 activity by tubacin or RNAi increased the level of MT acetylation.The addition of tubacin or down-regulation of HDAC6 significantly ameliorated the decreased migration and invasion phenotype induced by CAMSAP2 knockdown.Further,IF showed that inactivation or down-reuglation of HDAC6 reversed the structural and morphological changes induced by the down-regulation of CAMSAP2.To determine the effect of HDAC6 on CAMSAP2-mediated metastasis in vivo,cells were injected into the livers of nude mice toestablish an orthotopic metastatic model.Consistent with our in vitro results,HDAC6down-regulation markedly increased the incidence of lung metastasis and decreased the OS time of the MHCC97H-shCAMSAP2 group.We further evaluated the possible relationship between CAMSAP2 and either acetylated MTs or HDAC6 expression in HCC sample.IHC showed that CAMSAP2 overexpression positively correlated with acetylated ?-tubulin but inversely correlated with HDAC6 expression.Both Western blotting and IF confirmed these result.Further analysis of the clinicopathological characteristics in paired HCC tissues showed that either up-regulation of acetylated MTs or down-regulation of HDAC6 significantly correlated with malignant tumor progression and poor prognosis.Kaplan-Meier analysis showed that patients with positive co-expression of CAMSAP2 and acetylated ?-tubulin endured the highest recurrence rates and lowest OS times,meanwhile the CAMSAP2(+)/HDAC6(-)expression pattern indicated a poor prognosis.Part ?: Western blotting showed that CAMSAP2 down-regulation decreased the expression of phosphorylated JNK,phosphorylated c-Jun and acetylated MTs and increased HDAC6 protein levels in HCC cells.Next,we used SP600125,a potent JNK inhibitor,or c-Jun knockdown(denoted “Lenti-shc-Jun”)lentivirus to inactivate c-Jun in CAMSAP2-overexpressing HCC cells.Both pretreatment with SP600125 and c-Jun knockdown significantly ameliorated the decreased expression of HDAC6 induced by CAMSAP2 overexpression,and the level of ?-tubulin acetylation decreased markedly compared with that in the control CAMSAP2 group.Transwell and wound-healing assays revealed that JNK/c-Jun inactivation markedly suppressed the increased migration and invasion capabilities induced by CAMSAP2 up-regulation.These data suggest that activated c-Jun induces,through the up-regulation of CAMSAP2,repression of HDAC6 expression.CONCLUSIONS: In conclusion,this study reports a novel function of CAMSAP2 in HCC.CAMSAP2 promotes HCC invasion and metastasis by regulating noncentrosomal MT rearrangement and stabilizing acetylated MTs.Thus,CAMSAP2 may serve as a candidatebiomarker for HCC prognosis and as a potential target for therapeutic intervention.