Wong Carmen Chak-Lui, Wong Chun-Ming, Ko Frankie Chi-Fat, Chan Lo-Kong, Ching Yick-Pang, Yam Judy Wai-Ping, Ng Irene Oi-lin
Department of Pathology, SH Ho Foundation Research Laboratory and Jockey Club Clinical Research Centre, The University of Hong Kong, Hong Kong, China.
PLoS One. 2008 Jul 23;3(7):e2779. doi: 10.1371/journal.pone.0002779.
Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC).
METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immunofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage.
Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.
肝癌缺失基因1（DLC1）是RhoGTP酶激活蛋白（GAP）家族的成员，已知其在肿瘤发生和癌症转移中具有抑制活性。然而，DLC1抑制细胞运动的潜在分子机制尚未完全阐明。Rho激酶（ROCK）是RhoA在介导细胞细胞骨架事件和细胞运动中的直接下游效应物。在本研究中，我们旨在研究DLC1对肝细胞癌（HCC）中Rho/ROCK信号通路的影响。
方法/主要发现：我们证明DLC1负向调节ROCK依赖的肌动球蛋白收缩性。通过免疫荧光研究，我们发现DLC1的异位表达消除了Rho/ROCK介导的细胞骨架重组，包括应力纤维和粘着斑的形成。它还下调了肌球蛋白轻链2（MLC2）的皮质磷酸化。DLC1的这些抑制作用是RhoGAP依赖性的，因为DLC1的RhoGAP缺陷突变体（DLC1 K714E）消除了这些抑制作用。此外，通过蛋白质印迹研究，DLC1抑制了ROCK相关的肌球蛋白轻链磷酸酶靶向单位1（MYPT1）在苏氨酸853处的磷酸化。通过显微镜检查细胞形态，我们发现显性激活的ROCK的异位表达使细胞从DLC1诱导的细胞骨架塌陷和细胞收缩中释放出来。
我们的数据表明DLC1负向调节Rho/ROCK/MLC2。这暗示了DLC1通过ROCK介导的途径抑制HCC细胞的转移，并丰富了我们对肝细胞癌进展中涉及的分子机制的理解。
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