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).

中文翻译：

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肌动蛋白细胞骨架重塑驱动上皮间质转化为肝癌侵袭和转移

高侵袭性是人类肝细胞癌 (HCC) 的标志。大肿瘤预测侵袭和转移。上皮间质转化（EMT）对于癌症的侵袭和转移至关重要。然而，大肿瘤倾向于接受 EMT 的机制仍不清楚。我们进行了亚基因组范围的筛选，并通过抑制 EMT 将 KLHL23 鉴定为 HCC 侵袭抑制因子。KLHL23 与肌动蛋白结合并抑制肌动蛋白聚合。KLHL23 沉默诱导filopodium 和lamellipodium 形成。此外，KLHL23 通过其对肌动蛋白动力学的作用抑制了 EMT。传统上，肌动蛋白细胞骨架重塑是 EMT 重编程的下游。因此，有趣的是询问 KLHL23 为什么以及如何反向调节 EMT。通过 KLHL23 沉默或用肌动蛋白细胞骨架调节剂处理激活肌动蛋白细胞骨架重塑，以细胞密度依赖性方式增强细胞缺氧反应，导致缺氧诱导因子 (HIF) 和 Notch 信号以及随后的 EMT。除非同时激活肌动蛋白细胞骨架重塑，并且仅当细胞处于高密度时，否则环境缺氧不会诱导 EMT。由此产生的 EMT 被 5'-三磷酸腺苷补充剂或肌动蛋白聚合抑制剂逆转。KLHL23 的下调与 HCC 和胰腺癌的侵袭、转移和不良预后相关。肿瘤大小与 EMT 的相关性以及 KLHL23 表达与 HIF/Notch 信号的负相关在小鼠的患者来源的异种移植 HCC 中得到进一步验证。结论：通过内在（例如 KLHL23 下调）或微环境线索同时激活肌动蛋白细胞骨架重塑对于细胞密度依赖性和缺氧介导的 EMT 至关重要，提供了大肿瘤大小和侵袭/转移之间的机制联系。我们的发现为制定肿瘤侵袭和转移的预防和治疗策略提供了一种手段。（肝病学 2018 年；67：2226-2243）。