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Deleted in liver cancer 1 (DLC1) negatively regulates Rho/ROCK/MLC pathway in hepatocellular carcinoma
Carmen Chak-Lui Wong1, Chun-Ming Wong, Frankie Chi-Fat Ko
1Department of Pathology, SH Ho Foundation Research Laboratory and Jockey Club Clinical Research Centre, The University of Hong Kong, Hong Kong, China.
Aims:
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.
Conclusion:
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.
Insights
Deleted in liver cancer 1 (DLC1) suppresses hepatocellular carcinoma (HCC) cell motility by inhibiting the Rho-kinase (ROCK) pathway. This finding clarifies DLC1
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Deleted in liver cancer 1 (DLC1) is a RhoGTPase activating protein (GAP) family member with known tumor suppressive roles.
- The precise molecular mechanisms by which DLC1 inhibits cancer cell motility, particularly in hepatocellular carcinoma (HCC), remain incompletely understood.
- Rho-kinase (ROCK) is a key downstream mediator of RhoA signaling, regulating cytoskeletal dynamics and cell motility.
Purpose of the Study:
- To investigate the effect of DLC1 on the Rho/ROCK signaling pathway in HCC.
- To elucidate the molecular mechanisms underlying DLC1's suppression of HCC cell motility.
Main Methods:
- Ectopic expression of DLC1 and its RhoGAP-deficient mutant (DLC1 K714E) in HCC cells.
- Immunofluorescence microscopy to assess cytoskeletal organization (stress fibers, focal adhesions) and myosin light chain 2 (MLC2) phosphorylation.
- Western blotting to analyze phosphorylation of myosin light chain phosphatase targeting unit 1 (MYPT1).
- Microscopy to observe cell morphology following expression of dominant-active ROCK.
Main Results:
- DLC1 negatively regulates ROCK-dependent actomyosin contractility.
- Ectopic DLC1 expression abrogates Rho/ROCK-mediated cytoskeletal reorganization, including stress fiber and focal adhesion formation.
- DLC1 downregulates cortical MLC2 phosphorylation in a RhoGAP-dependent manner.
- DLC1 inhibits MYPT1 phosphorylation at Threonine 853.
- Overexpression of dominant-active ROCK rescues cells from DLC1-induced cytoskeletal collapse and shrinkage.
Conclusions:
- DLC1 negatively regulates the Rho/ROCK/MLC2 signaling pathway.
- This study implicates a ROCK-mediated mechanism for DLC1's suppression of HCC cell metastasis.
- The findings enhance the understanding of molecular mechanisms involved in HCC progression.
