Plexin B1 is repressed by oncogenic B-Raf signaling and functions as a tumor suppressor in melanoma cells

Abstract

Human melanomas show oncogenic B-Raf mutations, which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell-cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by mitogen-activated protein kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1, which mediate receptor GTPase activating protein activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor-suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor-suppressor function in early-stage cells, although suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.

Figure 1. Axon guidance genes are repressed by B-Raf/MKK/ERK in melanoma

(A, B) Summary of pathways containing genes significantly (A) decreased or (B) increased in response to treatment of eight melanoma cell lines with U0126. Significant responses are filtered by rank products and gene lists were sorted into KEGG pathway groups, listing those with summed weighted gene counts >2.5%. Genes decreased by U0126 are enriched in pathways required for cell cycle progression. Genes increased by U0126 are highest in axon guidance pathways. (C) Two-sample comparison of pathways that best discriminate between genes induced versus repressed by B-Raf/MKK/ERK. For each pathway, filled bars represent genes increased or decreased by U0126, while open bars represent genes respectively decreased or increased by U0126. For gene sets decreased by U0126, filled bars represent genes decreased and open bars represent genes increased by U0126. Asterisks indicate pathways that discriminate between with P-value<0.01. (D) Genes within the axon guidance gene group. Genes within other groups in panel C are presented in Suppl. Table 4.

Figure 2. Plexin B1 is repressed by B-Raf/MKK/ERK in melanoma cells and melanocytes

(A) RMA-normalized intensities for plexin B1 (probeset 215807_s_at) shows induction by U0126 (10 μM, 24 h) in 7 of 8 melanoma cell lines. (B) Western blots confirm induction of plexin B1 with 24 h treatment with U0126 (10 μM) or PD184352 (3 μM) in melanoma cell lines. (C) A time course in WM239A cells shows induction of plexin B1 by 12 h of U0126 (10 μM) treatment. (D) Dose response study in WM239A cells shows that plexin B1 induction is correlated with phospho-ERK1/2 inhibition by U0126 (24 h). (E) Reciprocal control by MKK/ERK is revealed in melanocytes, where plexin B1 is repressed upon adenoviral delivery of constitutively active MKK1. (F) Western blots show a modest induction of R-Ras by U0126, in 4 of 5 cell lines. (G) Western blots reveal the presence of semaphorin 4D in WM115 and WM239A cells, which is unaffected by treatment with U0126 (10 μM, 24 h). Equal amounts of lysates (20 μg) were loaded in all lanes.

Figure 3. Plexin B1 inhibits colony growth in soft agar

(A) WM115 and (B) WM239A cell lines expressing plexin B1 were resuspended in agar. Colony forming efficiency was measured after 21 days, measuring the ratio of colonies with diameter >200 μm to the total number of cells plated. Colonies formed in control lines are significantly reduced upon stable expression of plexin B1. Mutant plexin B1-RM, defective in R-Ras GAP activity, does not repress colony growth from melanoma cells. (C) Stable expression of plexin B1 has little effect on proliferation of WM115 or WM239A cells grown on 2D culture dishes. (D) Expression of GFP-PDZ in WM239A cells does not reverse the inhibitory response to plexin B1. Western blots showing expression of plexin B1-RM and GFP-PDZ are shown in Suppl. Fig. 3.

Figure 4. Plexin B1 inhibits spheroid growth in extracellular matrix

(A) WM115 and (B) WM239A cell lines were grown in collagen matrix for 7-10 days. Plexin B1 inhibits the formation of spheroids in both cell types, although small spheroid clusters are still apparent in WM239A cells, suggesting only partial inhibition in this cell line. In both cell types, formation of large spheroids is recovered by expression of the R-RasGAP defective mutant, plexin B1-RM. (C) WM115 and WM239A cells are cultured as spheroids in the presence of U0126 (10 μM) for 10 days, and then stained for cell viability, where green indicates live cells and red indicates dead cells. Neither U0126 treatment nor plexin B1 expression affected viability of spheroid cells. (D) WM239A cells expressing plexin B1, and either GFP (virus control) or GFP-PDZ were cultured as spheroids and implanted into collagen. Inhibiting plexin B1 activation of RhoA by expressing GFP-PDZ did not rescue spheroid growth, demonstrating that plexin B1 does not signal through RhoA.

Figure 5. Plexin B1 is a tumor suppressor in melanoma cells

(A) Xenograft tumors from WM115 cells are smaller and unpigmented, whereas xenografts from WM239A cells are larger and pigmented. No differences in tumor morphology due to plexin B1 expression are apparent in histological sections (hematoxylin + eosin). (B) Xenograft tumor growth from WM115 cells injected intradermally into athymic nude mice is strongly inhibited in cells stably expressing plexin B1. Similar results were obtained in cells injected with Matrigel. (C) In contrast, plexin B1 expression does not affect xenograft growth from WM239A cells.

Figure 6. Tumor suppressor activity of plexin B1 is correlated with repression of phospho-AKT

(A) Western blots show that phospho-AKT is repressed in WM115 cells stably expressing plexin B1, whereas no effect is observed in WM239A cells. Two biological replicates are shown for each condition. (B) Immunohistochemistry of xenografts from WM115 cells shows that phospho-AKT is significantly repressed in tumors from cells expressing plexin B1, whereas plexin B1 has no effect on phospho-AKT in WM239A xenografts. Each image is representative of tumors from three mice. (C) Western blots of lysates from xenografts of WM115 and WM239A cells show comparable levels of phospho-AKT and total AKT, normalized to TATA binding protein (TBP). Thus, the tumor suppressive effect of plexin B1 is correlated with its inhibition of phospho-AKT.

Figure 7. Plexin B1 inhibits migration in metastatic cells

(A) U0126 inhibits transwell cell migration in WM239A and WM115 cells, indicating that migration is positively regulated by B-Raf/MKK/ERK. (B) Stable expression of plexin B1 inhibits migration of WM239A cells, mimicking the effect of U0126. In contrast, migration of WM115 cells is not inhibited, and is somewhat elevated, by plexin B1.