The frequent mutational activation of B-RAF in melanoma coupled with the growing number of studies demonstrating its significant role in melanoma initiation and progression has validated B-RAF as an important therapeutic target. Although clinical grade RAF inhibitors have been developed (43
), their effective use to date has been diminished due to pharmacokinetic and specificity concerns (44
). In addition, recent evidence suggests that increased C-RAF expression may provide for a mechanism of acquired drug resistance (45
). Our studies have focused on delineating mutant B-RAF regulated targets involved in malignant traits. Complicating the analysis of these targets is the varying cellular contexts used. In this study, we analyzed the functional role of the B-RAF regulated GTPase Rnd3 in invasive melanoma cells. For the first time, our study addresses three critical issues pertaining to the role of Rnd3 in melanoma. Initially, we show that inducible knockdown of endogenous Rnd3 regulates actin cytoskeletal organization in a manner dependent on RhoA but not RhoB or RhoC. Next, we show that Rnd3 expression is elevated in melanoma cells that display invasive outgrowth from tumor spheroids. Finally, silencing of Rnd3 profoundly reduced the invasive component of spheroid outgrowth in 3-D collagen gels and effectively blocked collective and border cell movements on 3-D. Thus, the present work advances our knowledge of the signaling pathways influenced by Rnd3 and establishes its expression as a crucial regulator of invasive melanoma cytoskeletal organization and cell migration (Supplemental Fig. S5
Oncogene-induced alterations in the actin cytoskeleton are observed in a variety of cancerous cell types (21
). Here, we show that depletion of endogenous Rnd3 promotes an increase in actin stress fibers, consistent with findings in osteosarcoma cells (32
). Actin stress fiber formation results from activation of Rho GTPases and their subsequent effects on ROCK1/2 signaling (14
). Rnd3 was previously shown to inhibit Rho/ROCK signaling through its interaction with p190RhoGAP (47
) and ROCK1 (30
). However, Rnd3 was recently shown to disassemble stress fibers independent of ROCK1 binding (49
), which suggests that the Rnd3-p190RhoGAP interaction facilitates cytoskeletal disassembly. The RhoGAP domain of p190 RhoGAP can catalyze GTP hydrolysis of RhoA, RhoB and RhoC (50
). Currently, there is limited information addressing a potential isoform-specific role(s) for Rnd3 regulation of RhoA/B/C. Moreover, RhoA/B/C can all mediate stress fiber formation (26
) and all are expressed in melanoma. The current manuscript is the first to directly examine the contribution of RhoA/B/C isoforms to Rnd3-mediated cytoskeletal effects at the molecular level. Our results demonstrate that Rnd3 preferentially restricts RhoA signaling to regulate actin organization. This is consistent with a previous report in breast carcinoma demonstrating that the roles of RhoA/B/C are not redundant (17
). While our studies in VGP melanoma cells implicate inhibition of RhoA signaling in the control of actin stress fibers and cell migration, we cannot rule out a requirement for additional Rho isoforms during later stages of melanoma progression.
In non-melanoma cell types, Rnd3 expression has been linked to either increased or decreased cell proliferation (29
) and pro-survival effects (32
). In invasive melanoma which express mutant B-RAF, we did not observe changes in the cell cycle profile or growth following Rnd3 knockdown. Furthermore, the levels of ERK1/2 and AKT phosphorylation that regulate melanoma proliferation and survival, as well as cyclin D1 expression were similar between the control and Rnd3 depleted melanoma cell lines. Thus, it seems that the relationship between Rnd3 expression and cell proliferation varies between different types of cells. One explanation for these discrepancies may be that mutant B-RAF signaling in melanoma overrides any change in cell growth imposed by Rnd3.
The seemingly contradictory studies on Rnd3 expression in cancer (19
) as well as our previous results showing induction of Rnd3 by mutant B-RAF (21
), prompted us to examine the expression of Rnd3 in cells characteristic of non-invasive and invasive stages of melanoma. Data presented here show that increased Rnd3 expression correlated with the transition from non-invasive to invasive melanoma. Interestingly SBcl2 and WM35 cells, which harbor mutant N-RAS and mutant B-RAF, respectively, displayed low Rnd3 expression. This is likely due to the reduced phosphorylation of active ERK1/2 in SBcl2 and WM35. Differences in ERK1/2 activation in melanoma cells which harbor mutant B-RAF may be due to amplification of mutant B-RAF alleles in invasive cells or the presence of a negative feedback loop in non-invasive cells (6
). The mechanism of ERK1/2 regulation of Rnd3 in melanoma is largely unknown. Rnd3 has recently been reported to be a p53 transcriptional target gene (32
); however, knockdown of wild-type p53 in WM793 cells did not disrupt Rnd3 expression (Supplemental Fig. S6
). Now that a role for Rnd3 in invasive melanoma has been established, additional studies are warranted to identify the mechanism(s) responsible for ERK1/2 control of Rnd3 expression. In sum, it appears that Rnd3 expression in melanoma is associated with elevated B-RAF/MEK/ERK signaling, as well as a pro-invasive phenotype.
VGP melanoma is distinguished from RGP melanoma by its invasive movement into the dermis (1
). In a 3-D model system, we demonstrate that knockdown of endogenous Rnd3 dramatically attenuates the ability of spheroid cells to invade out into the surrounding collagen gel. Notably, the spheroid core increased in size and was composed of live cells, suggesting that overall growth was maintained. Analysis of spheroids placed on top of collagen gels revealed that migration of collective cell layers, as well as individual border cells was reduced in Rnd3-depleted spheroids. Although a precise mechanism for reduced migration in 3-D matrices is currently under investigation, the results from 2-D assays implicate deregulated RhoA signaling. This would be consistent with current reports implicating Rho/ROCK signaling in the regulation of tissue compliance and tumor cell migration (10
). Future studies will examine whether Rnd3 regulation of RhoA facilitates melanoma migration in 3-D matrices through its control of focal adhesion dynamics, extracellular matrix protease activity and/or cellular tension.
In conclusion, the current data advance our knowledge of Rnd3 and Rho GTPases in melanoma. Increased expression of Rnd3 attenuates RhoA-ROCK signaling in invasive melanoma to regulate 3-D migration. Our results potentially serve as the framework for new therapeutic strategies that target oncogene-regulated effector pathways to reduce invasive behavior and restrict malignancy in human cancer.