Lo Kong Chan

Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis†

Caveolin‐1 (Cav1) has been implicated in diverse human cancers, yet its role in hepatocellular carcinoma (HCC) tumourigenesis and metastasis remains elusive. In the current study, we aim to provide a comprehensive understanding regarding the functional role of Cav1 in HCC tumourigenesis and metastasis. Cav1 expression was examined in a panel of human HCC cell lines using western blotting analysis and quantitative RT‐PCR and human tissues by immunohistochemistry. Cav1 was not detected in normal liver cell line and all non‐tumourous liver tissues but exclusively expressed in HCC cell lines and tissues. Dramatic expression of Cav1 was found in metastatic HCC cell lines and tumours, indicating a progressive increase of Cav1 expression along disease progression. Cav1 overexpression was significantly correlated with venous invasion (p = 0.036). To investigate the functions of Cav1 in HCC, Cav1 overexpressing and knockdown stable clones were established in HCC cells and their tumourigenicity and metastatic potential were examined. Overexpression of Cav1 promoted HCC cell growth, motility, and invasiveness, as well as tumourigenicity in vivo. Conversely, knockdown of Cav1 in metastatic HCC cells inhibited the motility and invasiveness and markedly suppressed the tumour growth and metastatic potential in vivo. Collectively, our findings have shown the exclusive expression of Cav1 in HCC cell lines and clinical samples and revealed an up‐regulation of Cav1 along HCC progression. The definitive role of Cav1 in promoting HCC tumourigenesis was demonstrated, and we have shown for the first time in a mouse model that Cav1 promotes HCC metastasis. Copyright

Down‐regulation of TIMP2 by HIF‐1α/miR‐210/HIF‐3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma

Cancer metastasis is a multistep process that involves a series of tumor‐stromal interaction, including extracellular matrix (ECM) remodeling, which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases (TIMP) 2 in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly down‐regulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.8%; 23 of 55) in human HCCs and was significantly associated with liver invasion and poorer survival outcomes of HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced cell invasive ability and ECM degradation associated with formation of invadopodia‐like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Using an orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line, MHCC‐97L, significantly reduced HCC progression as well as pulmonary metastasis. Mechanistically, TIMP2 suppression, in a hypoxic environment, was induced through a regulatory feedback circuit consisting of hypoxia‐inducible factor (HIF) 1 alpha, microRNA‐210 (miR‐210), and HIF‐3α. Conclusion: TIMP2 is frequently down‐regulated in human HCCs and its down‐regulation is associated with aggressive tumor behavior and poorer patient outcome. Its suppression is under the regulation of a novel feedback circuit consisting of HIF‐1α/miR‐210/HIF‐3α. TIMP2 is an important regulator of ECM degradation and HCC metastasis. (Hepatology 2016;64:473‐487)

Akt Phosphorylation of Deleted in Liver Cancer 1 Abrogates Its Suppression of Liver Cancer Tumorigenesis and Metastasis

BACKGROUND & AIMSnDeleted in liver cancer 1 (DLC1), which encodes a Rho GTPase activating protein, is a bona fide tumor suppressor in hepatocellular carcinoma. Underexpression of DLC1 in cancer has been attributed to genomic deletion and epigenetic silencing. However, the regulatory mechanism of the tumor suppressive activity of DLC1 remains elusive. In this study, we elucidated a novel post-translational modification by which the activity of DLC1 is functionally regulated.nnnMETHODSnMolecular and biochemical approaches were employed to study Akt phosphorylation of DLC1. In vitro and in vivo functional assays were performed to elucidate the functional significance of Akt phosphorylation of DLC1.nnnRESULTSnPhosphorylation of ectopically expressed and endogenous DLC1 was enhanced upon insulin induction or with Akt expression in liver cancer cell lines. Conversely, addition of a phosphatidylinositol 3-kinase/Akt pathway inhibitor or silencing of Akt attenuated the phosphorylation level of DLC1. Site-directed mutagenesis was employed to replace the serine residue of the consensus Akt substrate motifs of DLC1 with alanine. S567 of DLC1 was identified as the only target of Akt phosphorylation. S567 is well conserved in all DLC family members. DLC2 was phosphorylated by Akt at the corresponding residue. Functional assays demonstrated that the S567D phosphomimetic DLC1 mutant lost its inhibitory activities in tumorigenesis and metastasis of oncogenically transformed hepatoblasts in a mouse model.nnnCONCLUSIONSnThis study has revealed a novel post-translational modification that functionally deregulates the biologic activities of DLC1. Phosphorylation of DLC1 and DLC2 by Akt at the conserved residue points to a common regulatory mechanism of the DLC tumor suppressor family.

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf-1-ERK1/2-p70S6K signalling pathway.

Background: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase‐activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation.

RhoE/ROCK2 regulates chemoresistance through NF-κB/IL-6/ STAT3 signaling in hepatocellular carcinoma

Small Rho GTPase (Rho) and its immediate effector Rho kinase (ROCK) are reported to regulate cell survival, but the detailed molecular mechanism remains largely unknown. We had previously shown that Rho/ROCK signaling was highly activated in hepatocellular carcinoma (HCC). In this study, we further demonstrated that downregulation of RhoE, a RhoA antagonist, and upregulation of ROCK enhanced resistance to chemotherapy in HCC in both in vitro cell and in vivo murine xenograft models, whereas a ROCK inhibitor was able to profoundly sensitize HCC tumors to cisplatin treatment. Specifically, the ROCK2 isoform but not ROCK1 maintained the chemoresistance in HCC cells. Mechanistically, we demonstrated that activation of ROCK2 enhanced the phosphorylation of JAK2 and STAT3 through increased expression of IL-6 and the IL-6 receptor complex. We also identified IKKβ as the direct downstream target of Rho/ROCK, and activation of ROCK2 significantly augmented NF-κB transcription activity and induced IL-6 expression. These data indicate that Rho/ROCK signaling activates a positive feedback loop of IKKβ/NF-κB/IL-6/STAT3 which confers chemoresistance to HCC cells and is a potential molecular target for HCC therapy.

Abstract 689: Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma

Cancer metastasis is a multi-step process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases 2 (TIMP2) in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly downregulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.1%; 23/56) in human HCCs as compared to the corresponding non-tumorous livers and was significantly associated with direct liver invasion into the adjacent liver parenchyma and poorer survival outcomes of the HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced the cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Furthermore, using orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line MHCC-97L not only significantly reduced the incidence of tumor microsatellite formation and venous invasion in the primary hepatic tumor xenografts, but also pulmonary metastasis, suggesting that both extrahepatic and intrahepatic metastasis in HCC was suppressed by TIMP2 expression. Mechanistically, TIMP2 suppression in a hypoxic environment was induced through a regulatory feedback circuit consisting of HIF-1a, miR-210 and HIF-3a. Taken altogether, our findings established that TIMP2 was frequently downregulated in human HCCs and its downregulation was associated with aggressive behavior and poorer patients’ outcome. Its suppression was under the regulation of a novel feedback circuit consisting of HIF-1a/ miR-210/ HIF-3a. Overall, our study has provided solid in vitro and in vivo evidence that TIMP2 is an important regulator of ECM degradation and HCC metastasis. These findings demonstrated that perturbation of the dynamic HIF-1a signaling circuit by miR-210 inhibitor abolished TIMP2 downregulation and suppressed HCC cell invasion. They also support the notion that targeting against HIF-1a signaling is a promising direction to tackle the hypoxic responses in HCC elicited by transarterial chemo-embolization (TACE) treatment to HCC patients. Citation Format: Alan KL Kai, Lo Kong Chan, Regina CL Lo, Joyce Lee, Carmen CL Wong, Jack CM Wong, Irene OL Ng. Downregulation of TIMP2 via HIF-1a/miR-210/HIF-3a regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 689.

Abstract 5292: The functional role of Tensin4 in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnThe focal adhesion protein Tensin4 (also known as cten: c-terminal tensin like) is structurally distinct from the three other members in the Tensin family and has been reported to play critical roles in cancer cell migration and invasion in breast and colorectal cancers. However, its expression and potential function in other cancers including hepatocellular carcinoma are not well understood. By quantitative real time PCR and western blotting with Tensin4 specific antibodies, Tensin4 was found to be expressed in a panel of HCC cell lines with a number of cells showing high levels of expression. In these cells, treatment with growth factors (e.g. EGF) significantly induced Tensin4 protein expression; while treatment of pharmacological inhibitors against the kinases in the MAPK signaling cascade abolished this induction. These observations suggested that Tensin4 expression was Ras/MAPK signaling dependent. Intriguingly, the growth factor-induced Tensin4 expression could not be observed in HCC cell lines with low basal Tensin4 expression, implying that other regulatory mechanisms do exist. By immunofluorescence microscopy, the focal adhesion localization of Tensin4 was confirmed in HCC cells. Detail examination of a panel of Tensin4 deletion constructs revealed the requirement of the C-terminal SH2 domain for this specific localization. To examine the functional role of Tensin4, SMMC and BEL7402 HCC cells with high basal expression of Tensin4 were subjected to stable Tensin4 knockdown followed by cell proliferation and cell migration assays. Consistently, we observed that both stable Tensin4 knockdown cells proliferated and migrated in slower rates in vitro. However, the molecular mechanisms regarding this reduction remains to be explored. Taken together, our data suggested that Tensin4 may play a pro-oncogenic role in HCC, possibly function as a downstream effector of Ras/MAPK signaling.nnThe study was funded by a Hong Kong Research Grants Council GRF grant (775110).nnCitation Format: Lo Kong Chan. The functional role of Tensin4 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5292. doi:10.1158/1538-7445.AM2014-5292

Abstract 2141: cAMP/PKA signaling enhances activity of Deleted in Liver Cancer 1 (DLC1) tumor suppressor in suppressing liver cancer tumorigenesis

Deleted in Liver Cancer 1 (DLC1) which encodes a RhoGTPase-activating protein (RhoGAP) is a tumor suppressor frequently inactivated in a wide spectrum of human cancers. The RhoGAP activity has been shown to play a predominant role in the biological activities of DLC1. It has been shown that cells with silenced DLC1 exhibit increased active RhoA level. This finding provides evidence about the activation of RhoA as the consequence of deregulated DLC1 and points to the importance of RhoGAP activity in the biological activity of DLC1. In this regard, it is important to comprehend how RhoGAP activity of DLC1 is related. Here, we show that DLC1 was robustly phosphorylated by cyclic AMP (cAMP)/Protein kinase A (PKA) signaling in cells. Phosphorylation of DLC1 was enhanced by forskolin, a known activator of PKA while suppressed when H-89, an inhibitor of PKA was added. Direct phosphorylation of DLC1 by PKA was further confirmed by the in vitro kinase assay. Using specific phospho-DLC1 antibodies, PKA was shown to phosphorylate DLC1 at S431 and S549. Functional assays demonstrate that phosphorylation at S549 plays a critical role in provoking the inhibitory activity of DLC1 in suppressing growth and motility of Ras-transduced p53 null mouse hepatoblasts. When compared with the stable clone of wild-type DLC1, stable clone of DLC1 phosphomimetic mutant, S549D, displayed a largely reduced growth of subcutaneous and orthotopic liver implanted tumors and an enhanced apoptosis. The migration and invasion rates of S549D cells were also significantly inhibited. Furthermore, S549D expression abolished stress fiber formation but failed to alter filopodia protrusions. These functional effects exerted by S549D were ascribed to the enhanced RhoGAP activity against RhoA. To further investigate the mechanism through which the RhoGAP activity is enhanced, we found that DLC1 dimerized upon S549 phosphorylation. Our findings have revealed for the first time about the regulation of RhoGAP activity of DLC1 via dimerization. Our study suggests a molecular link between PKA and DLC1/Rho pathways and underscores the importance of S549 phosphorylation in the regulation of RhoGAP activity of DLC1. (This study was funded by the Small Project Funding Program, The University of Hong Kong 200907176125) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2141. doi:1538-7445.AM2012-2141

Abstract 5007: Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnThe tumor suppressor Deleted in liver cancer 1 (DLC1) protects cell from transformation by catalyzing the GTP hydrolysis of active RhoA. Besides the RhoGAP activity, accumulating evidences have supported that localization at focal adhesions and interaction with tensin protein are key factors guiding the tumor suppressive activity of DLC1. In this study, we provided new evidence that DLC1 was a dynamic protein which shuttles between cytoplasm and nucleus instead of statically staying in the cytoplasm. Subcellular fractionation assay showed that DLC1 protein can be detected in cytoplasmic and nuclear fractions. Treatment of Leptomycin B (LMB), a nuclear export blocker, could lead to the nuclear retention of exogenous and endogenous DLC1 in different human cancer cell lines. Detail examination of relevant DLC1 mutants had shown that the center region of DLC1 was necessary and sufficient for its nuclear localization. We transiently expressed a NLS fusion DLC1 (NLS-DLC1) with preferential nuclear localization in SMMC HCC cells and found that NLS-DLC1 was less potent in suppressing colony formation and actin stress fiber formation. To study the tumor suppressive function of nuclear DLC1 both in vitro and in vivo, we employed retroviral transduction to stably express NLS-DLC1 in a p53−/− RasV12 hepatoblast model. We found that NLS-DLC1 expressing cells proliferated faster in vitro and exhibited increased tumorigenicity upon nude mice injection when compared with the non-nuclear targeted DLC1. Although the function of nuclear DLC1 remains to be answered, our results clearly demonstrated that nuclear localization of DLC1 may serve as a potential mechanism in negatively regulating its tumor suppressive activity.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5007.