A switch in RND3-RHOA signaling is critical for melanoma cell invasion following mutant-BRAF inhibition
© Klein and Higgins; licensee BioMed Central Ltd. 2011
Received: 25 March 2011
Accepted: 14 September 2011
Published: 14 September 2011
The initial use of BRAF targeted therapeutics in clinical trials has demonstrated encouraging responses in melanoma patients, although a rise in drug-resistant cells capable of advancing malignant disease has been described. The current study uses BRAFV600E expressing WM793 melanoma cells to derive data aimed at investigating the molecular determinant of cell invasion following treatment with clinical BRAF inhibitors.
Small-molecule inhibitors targeting BRAF reduced MEK1/2-ERK1/2 pathway activation and cell survival; yet, viable cell subpopulations persisted. The residual cells exhibited an elongated cell shape, prominent actin stress fibers and retained the ability to invade 3-D dermal-like microenvironments. BRAF inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling. Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival. The invasive ability of BRAF inhibitor treated cells embedded in collagen gels was diminished following RND3 re-expression or RHOA depletion. Conversely, melanoma cell movement in the absence of BRAF inhibition was unaffected by RND3 expression or RHOA depletion.
These data reveal a novel switch in the requirement for RND3 and RHOA in coordinating the movement of residual WM793 cells that are initially refractive to BRAF inhibitor therapy. These results have important clinical implications because they suggest that combining BRAF inhibitors with therapies that target the invasion of drug-resistant cells could aid in controlling disease relapse.
Cutaneous melanoma is the most lethal skin cancer and its incidence rates continues to rise . Clinical grade small molecule inhibitors targeting BRAF have recently emerged due to its frequent mutational status  and vital role in malignancy [3, 4]. In particular, a structure-based approach led to the development of PLX-4720, a potent inhibitor of BRAF kinase activity with a V600E mutation . PLX-4720 selectively inhibits MEK1/2-ERK1/2 activation, cell proliferation and xenograph tumor growth using mutant BRAF expressing cell lines [5, 6]. PLX-4720 is an analog of the clinically tested PLX-4032 (aka RG7204/Vermurafenib) compound which has demonstrated favorable therapeutic responses [7–9]. Although the durability of PLX-4032 is still under investigation, tumor relapse has been reported [7, 8].
A combination of strategies has been suggested to be required for successful therapeutic outcomes in melanoma [10, 11]. The addition of an anti-invasive agent to complement targeted BRAF inhibition constitutes an additional therapy that may improve patient outcomes by preventing or delaying the dissemination of drug-resistant clones; however, little is known regarding melanoma invasive strategies following BRAF inhibition. RND3-RHOA cell signaling was identified as a mutant-BRAF regulated pathway  that coordinates cell movement . RND3 is an atypical RHO-GTPase  that antagonizes RHO-ROCKI signaling [15, 16]. Whether this pathway participates in melanoma invasion following BRAF inhibition is unknown.
BRAF knockdown alters cytoskeletal architecture and cell shape ; therefore, it was important to assess whether alterations in F-actin also accompanied pharmaceutical BRAF inhibition. Control cells plated on collagen gels exhibited diffuse microfilament staining patterns with thin cortical fibers (Figure 2B). In contrast, prominent F-actin stress fibers typified BRAF inhibitor treated cells (Figure 2B); these stress fiber traversed the cell body often terminating in large bundles at the cell membrane. Cell elongation and prominent actin stress fibers, therefore, correlate with viable melanoma cells in the presence of BRAF inhibitors.
To determine if drug insensitivity occurred in a more physiological setting melanoma spheroids embedded into a 3-D collagen gel, to recapitulate a stromal-like environment , were treated with inhibitors in complete medium. Controls cultures invaded the surrounding extracellular matrix (Figure 2C). SB-590885 and PLX-4720 treatment attenuated invasive outgrowth (Figure 2C), although some spheroids were surrounded with elongated cells that invaded the surrounding microenvironment (Figure 2C). Invasive cells were evident in 33% and 36% of spheroid structures treated with SB-590885 and PLX-4720, respectively, (Figure 2D) clearly signifying that some cells can invade a 3-D microenvironment following pharmaceutical BRAF inhibition.
Cancer cell resistance to cytotoxic agents is a common and severe therapeutic impediment that can lead to the reemergence of malignant tumors. This study demonstrates that a subpopulation of melanoma cells can survive and invade a dermal-like extracellular matrix, despite BRAF inhibitor treatments. These findings agree with others who have shown that melanoma cell lines expressing a BRAFV600E mutation can established resistance to BRAF inhibitors in culture [20, 21] as well as a xenograph mouse model . Moreover, despite encouraging clinical trial outcomes using PLX-4032 [7, 8], the development of BRAF inhibitor resistant cells has been reported [23–25]. Collectively these studies advocate for the preparation of therapies that prevent the development of drug-insensitive clones or block the ability of these cells to spread and metastasize.
The present work identifies factors that facilitate the residual invasion of BRAFV600E expressing melanoma cells after pharmaceutical BRAF inhibition by employing 2-D and more physiological 3-D preclinical models. Initially, an elongated cell shape with prominent actin stress fibers were identified as phenotypic markers of viable cells following BRAF inhibition. Importantly, the correlation between cytoskeletal remodeling and drug insensitivity does not implicate prominent actin stress fibers as a predictive factor or "biomarker" for melanoma resistance to BRAF inhibition. The development of actin stress fibers more closely reflects enhanced RHOA pathway signaling. The current study identifies novel roles for RND3 and RHOA in the movement but not growth or survival of melanoma cells treated with BRAF inhibitors. These findings suggest that BRAF inhibition invokes a switch in the utilization of the RND3-RHOA signaling pathway. Accordingly, RND3 expression and suppressed RHOA signaling appear to be important for normal melanoma cell movement, whereas RND3 downregulation and enhanced RHOA signaling are critical in BRAF-inhibitor treated cells. Collectively, these data demonstrate that interfering with signaling pathways which facilitate the invasion of drug-resistant tumor cells may represents a cytostatic therapy that could complement BRAF inhibitor therapeutics.
List of abbreviations
RMK is Research Associate in the Center for Cell Biology and Cancer Research at Albany Medical College. PJH is Professor and Co-Director of the Center for Cell Biology and Cancer Research at Albany Medical College.
We thank Dr. Andrew Aplin for generously providing the WM793/TR/RND3 and WM793/TR/RHOA shRNA cell lines and reviewing this manuscript. We also thank Drs. J. Jordan, G. Liu and Mr. C. Higgins for technical assistance. This work was supported by grants from the American Cancer Society PF-08-032-01-CSM (RM Klein) and RSG-08-03-01-CSM (AE Aplin); National Institutes of Health R01-GM067893 (AE Aplin), R01-CA125103 (AE Aplin), R01-GM57242 (PJ Higgins) and ARRA-GM067893-S1 (AE Aplin); and Pennsylvania Department of Health AF0301 (AE Aplin).
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