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High invasiveness is a hallmark of human hepatocellular carcinoma (HCC). Large tumors predict invasion and metastasis. Epithelial-mesenchymal transition (EMT) is crucial for cancer invasion and metastasis. However, the mechanisms whereby large tumors tend to undergo EMT remain unclear. We conducted a subgenome-wide screen and identified KLHL23 as an HCC invasion suppressor by inhibiting EMT. KLHL23 binds to actin and suppresses actin polymerization. KLHL23 silencing induced filopodium and lamellipodium formation. Moreover, EMT was suppressed by KLHL23 through its action on actin dynamics. Traditionally, actin cytoskeleton remodeling is downstream of EMT reprogramming. It is therefore intriguing to ask why and how KLHL23 inversely regulates EMT. Activation of actin cytoskeleton remodeling by either KLHL23 silencing or treatment with actin cytoskeleton modulators augmented cellular hypoxic responses in a cell-density-dependent manner, resulting in hypoxia-inducible factor (HIF) and Notch signals and subsequent EMT. Environmental hypoxia did not induce EMT unless actin cytoskeleton remodeling was simultaneously activated and only when cells were at high density. The resulting EMT was reversed by either adenosine 5'-triphosphate supplementation or actin polymerization inhibitors. Down-regulation of KLHL23 was associated with invasion, metastasis, and poor prognosis of HCC and pancreatic cancer. Correlations of tumor size with EMT and inverse association of expression of KLHL23 with HIF/Notch signals were further validated in patient-derived xenograft HCCs in mice. Conclusion Simultaneously activation of actin cytoskeleton remodeling by intrinsic (such as KLHL23 down-regulation) or microenvironment cues is crucial for cell-density-dependent and hypoxia-mediated EMT, providing a mechanistic link between large tumor size and invasion/metastasis. Our findings provide a means of developing the prevention and treatment strategies for tumor invasion and metastasis. (Hepatology 2018;67:2226-2243).

It is unclear how proliferating cells elicit suppression on cell proliferation and how cancer cells evade this growth suppression. Using a loss-of-function screening of the human kinome and phosphatome to identify genes suppressing tumor initiation in human hepatocellular carcinoma (HCC), we identified 19 genes and characterized one of the top-scoring tumor suppressor candidates, protein tyrosine phosphatase receptor type F (PTPRF). We found that PTPRF was induced during cell proliferation by cell-cell contact. Ectopic expression of wild-type PTPRF, but not the phosphatase-inactive mutant, suppressed cell proliferation and colony formation in soft-agar assays. In contrast, PTPRF silencing led to cell hyperproliferation, enhanced tumor colony formation in soft agar, and increased xenograft tumor growth in nude mice. Mechanistically, PTPRF silencing showed aberrant ERK-dependent signaling including the phosphorylation/stabilization of v-myc avian myelocytomatosis viral oncogene homolog (MYC) through the direct activation of v-src avian sarcoma viral oncogene homolog (SRC) and suppression of PP2A. This PTPRF-mediated growth suppression during cell proliferation functioned independently of the Hippo-Yap pathway. Clinically, PTPRF was down-regulated in 42% HCC (37/89), 67% gastric cancer (27/40), and 100% colorectal cancer (40/40). PTPRF up-regulation was found in 24% HCC (21/89) and associated with better clinical outcomes. Conclusion: A novel PTPRF-mediated growth suppression pathway was identified by way of a functional genomics screening in human hepatoma cells. Induction of PTPRF by cell-cell contact during cell proliferation quenched the activated ERK-dependent proliferation signaling to prevent cell hyperproliferation and tumor initiation. PTPRF down-regulation in HCC facilitated tumor development. Our findings shed light on how cancer cells can evade growth suppression and open a new avenue for future development of anticancer therapies. (Hepatology 2014;59:2238–2250)

Intrahepatic metastasis is the primary cause of the high recurrence and poor prognosis of human hepatocellular carcinoma (HCC). However, neither its molecular mechanisms nor markers for its prediction before hepatectomy have been identified. We recently revealed up-regulation of erythroblastic leukemia viral oncogene homolog 3 (ERBB3) in human HCC. Here we examined the clinical and biological significance of ERBB3 in HCC. Up-regulation of ERBB3 in HCC was strongly associated with male gender (P< 0.001), chronic hepatitis B (P = 0.002), microscopic vascular invasion (P = 0.034), early recurrence (P = 0.003), and worse prognosis (P = 0.004). Phosphorylated ERBB3 and its ligands [neuregulins (NRGs)] were detected in both HCC tissues and cells. Phosphorylation of ERBB3 could be induced by conditioned media of HCC cells and abolished by the pretreatment of conditioned media with anti-NRG antibodies or by the silencing of the endogenous NRG expression of the donor HCC cells. Human epidermal growth factor receptor 2 was required for ERBB3 phosphorylation. The downstream phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene homolog pathways were primarily elicited by NRG1/ERBB3 signaling, whereas the mitogen-activated protein kinase/extracellular signal-regulated kinase pathways were elicited by both epidermal growth factor/epidermal growth factor receptor and NRG1/ERBB3 signaling. The activation and silencing of ERBB3-dependent signaling had potent effects on both the migration and invasion of HCC cells, but neither had significant effects on the proliferation of HCC cells, tumor formation, or tumor growth in vitro and in vivo. Conclusion: The constitutive activation of ERBB3-dependent signaling via the NRG1/ERBB3 autocrine loop plays a crucial role in the regulation of cell motility and invasion, which contribute to intrahepatic metastasis and early recurrence of HCC. ERBB3 is a marker for the prediction of intrahepatic metastasis and early recurrence. ERBB3-dependent signaling is a candidate target for the treatment of microscopic vascular invasion and for the prevention of HCC recurrence. (HEPATOLOGY 2011;53:504-516) (This article firstt published online on January 18, 2011; the title has since changed; the correct version appears in print.