Actin cytoskeleton remodeling drives epithelial-mesenchymal transition for hepatoma invasion and metastasis in mice.

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).

A novel interaction of nucleophosmin with BCL2-associated X protein regulating death evasion and drug sensitivity in human hepatoma cells.

UNLABELLED: Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently, we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells-particularly those bearing inactivated p53 gene (Huh7, Hep3B, and Mahlavu)-to ultraviolet irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not changed further, as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of the nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation revealed a direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, up-regulation of NPM was significantly associated with advanced tumor stage and poor prognosis. CONCLUSION: By directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential cotarget in combination with other therapies for HCC, particularly those that harbor inactivated p53 gene. Our findings are of clinical significance because NPM up-regulation and p53 mutations are usually found in advanced human cancers, including HCC.

Secreted ERBB3 isoforms are serum markers for early hepatoma in patients with chronic hepatitis and cirrhosis.

Most hepatocellular carcinoma (HCC) is generated from chronic hepatitis and cirrhosis. To discover new markers for early HCC in patients with chronic hepatitis and cirrhosis, we initiated our search in the interstitial fluid of tumor (TIF) via differential gel electrophoresis and antibody arrays and identified secreted ERBB3 isoforms (sERBB3). The performance of serum sERBB3 in diagnosis of HCC was analyzed using receiver operating characteristic curves (ROC). The serum sERBB3 level was significantly higher in HCC than in cirrhosis (p < 0.001) and chronic hepatitis (p < 0.001). The accuracy of serum sERBB3 in detection of HCC was further validated in two independent sets of patients. In discrimination of early HCC from chronic hepatitis or cirrhosis, serum sERBB3 had a better performance than alpha-fetoprotein (AFP) (areas under ROC [AUC]: sERBB3 vs AFP = 93.1 vs 81.0% from chronic hepatitis and 70.9 vs 62.7% from cirrhosis). Combination of sERBB3 and AFP further improved the accuracy in detection of early HCC from chronic hepatitis (AUC = 97.1%) or cirrhosis (AUC = 77.5%). Higher serum sERBB3 levels were associated with portal-vein invasion and extrahepatic metastasis of HCC (p = 0.017). Therefore, sERBB3 are serum markers for early HCC in patients with chronic hepatitis and cirrhosis.

Stathmin1 overexpression associated with polyploidy, tumor-cell invasion, early recurrence, and poor prognosis in human hepatoma.

Frequent intrahepatic metastasis causes early tumor recurrence and dismaying prognosis of human hepatocellular carcinoma (HCC). We recently identified overexpression of stathmin1 (STMN1) in human HCC. This study was designed to elucidate the clinical and biological significance of overexpression of STMN1 in HCC. Expression of STMN1 was conducted by quantitative reverse transcription-polymerase chain reaction and immunoblotting assays on 58 pairs of HCC and para-tumor liver tissues from patients with HCC along with normal liver tissues as the controls. Association of STMN1 overexpression with tumor recurrence and prognosis was investigated by Kaplan-Meier cumulative survival and Cox Regression analyses. Roles of STMN1 in cell cycle, cell motility, and invasion were determined by in vitro assays. STMN1 overexpression in hepatoma was strongly associated with local invasion (P = 0.031), early recurrence (P = 0.002), and poor prognosis (P = 0.005), and was an independent indicator for tumor recurrence (P = 0.0045). STMN1 overexpression further identified subgroups of HCC patients with higher tumor recurrence and worse prognosis among HCC patients with early tumor stage (T1) or intermediate histological grades (G2 and G3), both of whom represent the majority of HCC patients receiving primary curative hepatectomy. Silencing STMN1 expression via RNA interference suppressed invasion activity, while ectopic expression of STMN1 enhanced cell invasion and caused polyploidy of cells. In conclusion, STMN1 overexpression could predict early tumor recurrence and poor prognosis, particularly at early stage of hepatoma. Overexpression of STMN1 promoted polyploidy formation, tumor-cell invasion, and intrahepatic metastasis, suggesting that STMN1 can be a target for anti-cancer therapy of human hepatoma.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis.

AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn's disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (tau = 0.2145, P = 0.0281) and histologic grade (tau = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activation of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.