Mutant B-RAF regulates a Rac-dependent cadherin switch in melanoma

Abstract

The ability of cells to invade into the dermis is a critical event in the development of cutaneous melanoma and ultimately an indicator of poor prognosis. However, the molecular events surrounding the acquisition of this invasive phenotype remain incompletely understood. Mutations in B-RAF are frequent in melanoma and are known to regulate the invasive phenotype. In this study, we sought to determine the molecular mechanisms controlling melanoma invasion. We found that mutant B-RAF signaling regulates a cadherin switch. In melanoma cells expressing mutant B-RAF we observed high levels of N-cadherin and low levels of E-cadherin. Depletion of mutant B-RAF, by small interfering RNA, caused a decrease in the levels of N-cadherin and an increase in the levels of E-cadherin. Mechanistically, we found that this cadherin switch required the activity of Rac1 and its GEF, Tiam1, both of which show suppressed activity in the presence of mutant B-RAF. Consistent with the work of others, we found that depletion of mutant B-RAF decreased the invasive capacity of the melanoma cells. However, simultaneous depletion of B-RAF and Rac or Tiam1 resulted in invasive capacity similar to that of control cells. Taken together, our results suggest that mutant B-RAF signaling downregulates Tiam1/Rac activity resulting in an increase in N-cadherin levels and a decrease in E-cadherin levels and ultimately enhanced invasion.

Figure 1

BRAF knockdown causes a cadherin switch. A) WM793 cells were transfected with control, total B-RAF (BRAF) or mutant B-RAF (V600E) siRNA. Cell lysates were analyzed by Western blotting for BRAF, P-ERK, ERK, E-cadherin, N-cadherin and actin (as a loading control). B) WM793 cells were treated with5uM PLX4720 for 24 hours. N-cadherin and E-cadherin expression as well as P-Erk levels were analyzed by Western blotting. Actin was used as a loading control. C) WM793 cells were treated with 1uM U0126 for 24 hours. N-cadherin and E-cadherin expression as well as P-Erk levels were analyzed by Western blotting. Actin was used as a loading control. D) WM115 cells were transfected with control, total B-RAF (BRAF) siRNA or 1uM U0126. Cell lysates were analyzed by Western blotting for P-Erk, N-cadherin and E-cadherin levels. Actin was used as a loading control.

Figure 2

BRAF knockdown results in the expression of a functional E-cadherin. A) WM793 cells were transfected with BRAF siRNA for 72h. Cells were then stained for the presence of E-cadherin. B) WM793 cells were transfected with control or BRAF siRNA. Cells were then lysed with immunoprecipitation buffer and β-catenin was immunoprecipitated from cells. Immunoprecipitates were then blotted for the presence of E-cadherin. C) WM793 cells were transfected with control siRNA or BRAF siRNA and co-cultured with either MRC5 cells (fibroblasts) or HaCat cells (keratinocytes) on transwell filters. After 72h TER readings were taken with a voltohmeter. * indicates statistical significance when compared to control treatment (p<0.05, Student's t-test).

Figure 3

BRAF knockdown results in the activation of Rac1. A) WM793 cells were transfected with control, total B-RAF (BRAF) or mutant B-RAF (V600E) siRNA. The cells were lysed and the activation of Rac was measured through a GST-PBD pulldown assay as described in the Methods. B and C) WM793 cells were treated with 5uM PLX4720 (B) or 1uM U0126 (C) for 24 hours. The cells were lysed and the activation of Rac was measured through a GST-PBD pulldown assay as described in the Methods. D) WM115 cells were transfected with control, total B-RAF (BRAF) siRNA or 1uM U0126. The cells were lysed and the activation of Rac was measured through a GST-PBD pulldown assay as described in the Methods.

Figure 4

The cadherin switch induced upon BRAF knockdown requires Rac. A) WM793 cells were transfected with control, total B-RAF (BRAF) or mutant B-RAF (V600E) siRNA and Rac siRNA. Cell lysates were analyzed by Western blotting for E-cadherin, N-cadherin or actin (loading control). The levels of B-RAF and Rac were also analyzed to confirm knockdown. B) Cells were treated overnight with NSC. Cell lysates were analyzed by Western blotting for E-cadherin, N-cadherin or actin (loading control). To confirm the activity of the NSC compound cells were lysed and the activation of Rac was measured through a GST-PBD pulldown assay as described in the Methods. C) WM115 cells were transfected with control, total B-RAF (BRAF) siRNA and Rac siRNA. Cell lysates were analyzed by Western blotting for E-cadherin, N-cadherin or actin (loading control). The levels of B-RAF and Rac were also analyzed to confirm knockdown.

Figure 5

Melanocytes experience a cadherin switch upon mutant B-Raf expression. NHEM were transfected with B-Raf V600E for 24 hours. Cells were lysed and the lysates were analyzed for the presence of B-Raf, E-cadherin, N-cadherin, GTP-Rac, total Rac and actin (loading control).

Figure 6

The BRAF- induced cadherin switch does not occur in N-Ras mutant melanoma cells. A) VMM39, WM115 and WM793 cell lysates were blotted to compare relative levels of p-Erk, Rac and active Rac. Actin was used as a loading control. B) VMM39 cells were transfected with control siRNA, BRAF siRNA or treated with 1uM U0126. Cells were lysed and samples were blotted for N-cadherin, E-cadherin, active Rac, total Rac and p-Erk levels. Actin was used as a loading control.

Figure 7

BRAF knockdown activates Tiam1. WM793 cells were transfected with control, total B-RAF (BRAF) or mutant B-RAF (V600E) siRNA and Rac siRNA. Pulldowns were then performed with GST-Rac(15A) and samples blotted with antibodies against Tiam1 (A) and Vav2 (B). B) WM793 cells were transfected with control or BRAF siRNA. Cells were then lysed, scraped and lystates homogenized. The lysates were then centrifuged to separate cytolsolic and membrane fractions. The fractions were then analyzed by Western blotting for the Tiam1.

Figure 8

Rac activation observed upon BRAF knockdown requires Tiam1. WM793 (A) cells or WM115 cells (B) were transfected with control, Tiam1 (A, B) or Vav2 (C) siRNA in addition to BRAF or V600E siRNA. The cells were lysed and the activation of Rac was measured through a GST-PBD pulldown assay as described in the Methods. The levels of BRAF and Tiam1 (A,B) or Vav (C) were checked to confirm knockdown.

Figure 9

The cadherin switch observed upon BRAF knockdown requires Tiam1. WM793 cells (A) or WM115 cells (B) were transfected with control, BRAF, V600E and Tiam1 (A, B) or Vav2 (C) siRNA. N-cadherin and E-cadherin expression were analyzed by Western blotting. Actin was used as a loading control. The levels of BRAF and Tiam1 (A,B) or Vav (C) were checked to confirm knockdown.

Figure 10

BRAF knockdown regulates the invasive capacity of melanoma cells through Tiam1 and Rac. WM793 cells were transfected with control, BRAF, V600E and Rac (A), Tiam1 (B) or Vav2 (C) siRNA. A 24 hour Matrigel cell invasion assay was performed after knockdown. Asterisks denote statistical significance comparing knockdown cells with controls. D) WM793 cells were transfected with control, BRAF or V600E siRNA and proliferation was measured through an RTCA assay. * indicates statistical significance when compared to control treatment (p<0.05, Student's t-test).

Figure 10

BRAF knockdown regulates the invasive capacity of melanoma cells through Tiam1 and Rac. WM793 cells were transfected with control, BRAF, V600E and Rac (A), Tiam1 (B) or Vav2 (C) siRNA. A 24 hour Matrigel cell invasion assay was performed after knockdown. Asterisks denote statistical significance comparing knockdown cells with controls. D) WM793 cells were transfected with control, BRAF or V600E siRNA and proliferation was measured through an RTCA assay. * indicates statistical significance when compared to control treatment (p<0.05, Student's t-test).

Figure 11

Model of mutant BRAF Rac-dependent cadherin switch. In melanoma cells expressing BRAFV600E the activity of MEK and ERK is high, leading to low Tiam1 activity followed by low Rac activity. Low Rac activity results in high expression of N-cadherin and low expression of E-cadherin and enhanced invasiveness. (Black arrows outline the pathway. Red arrows show the relative activity level or expression of the indicated molecule).