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Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line
P M Ghosh1, N Ghosh-Choudhury, M L Moyer
1Department of Pathology, University of Texas Health Science Center, San Antonio 78284, USA.
Abstract:
Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.
Insights
Lovastatin halts prostate cancer cell growth by inhibiting RhoA. Restoring geranylgeranylation with geranylgeraniol rescues RhoA activity and cell proliferation, while farnesol promotes cell spreading via H-Ras but not proliferation.
Area of Science:
- Oncology
- Cell Biology
- Biochemistry
Background:
- Prostate cancer progression involves complex cellular signaling pathways.
- The HMG-CoA reductase inhibitor lovastatin affects cell growth and survival.
- RhoA, a small GTPase, plays a critical role in regulating cell cytoskeleton and proliferation.
Purpose of the Study:
- To investigate the role of RhoA in lovastatin-induced effects on prostate cancer cells.
- To determine the impact of geranylgeranylation and farnesylation on TRAMP cell behavior.
Main Methods:
- Treatment of TRAMP cells with lovastatin.
- Addition of geranylgeraniol (GGOL) and farnesol (FOL) to assess rescue effects.
- Analysis of RhoA activity, actin cytoskeleton, cell cycle, and apoptosis.
- Expression of dominant-negative and constitutively active RhoA mutants.
- Expression of dominant-negative H-Ras mutant.
Main Results:
- Lovastatin induced RhoA inactivation, cell cycle arrest, detachment, and apoptosis in TRAMP cells.
- Geranylgeraniol (GGOL) addition prevented lovastatin's effects by restoring RhoA activity and cell proliferation.
- Farnesol (FOL) addition prevented morphological changes and apoptosis but not growth arrest, suggesting a role for H-Ras in cell spreading.
- Dominant-negative RhoA expression slowed growth, while constitutively active RhoA accelerated it.
Conclusions:
- RhoA is essential for TRAMP cell proliferation and survival.
- Geranylgeranylation of RhoA is critical for maintaining TRAMP cell growth.
- H-Ras mediates cell spreading independently of RhoA geranylgeranylation, but cannot support proliferation without it.