WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

Abstract

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

Figure 1. WNT5A expression is increased in response to chronic inhibition of BRAF^V600E with PLX4720.

(A) Fold change in WNT5A mRNA expression normalized to GAPDH in BRAFi-resistant patient tumors (as marked by disease progression [prog]) compared with paired tumor biopsies collected before the start of treatment (pre). (B) Normalized viability of A375 and MEL624 parental and PLX-resistant cells (-R) after treatment with either medium, DMSO, or increasing doses of PLX4720 for 48 hours. Resistant cell lines (A375-R and MEL624-R) were derived by treatment of melanoma cells with 2 μM PLX4720 for more than 10 weeks. (C) Western blots of lysates from A375, A375-R, MEL624, and MEL624-R cells after either culturing in standard growth medium or treatment with vehicle control (DMSO) or 2 μM PLX4720 for 2 days. (D) Hierarchical clustering of gene expression in PLX-resistant (-R) and PLX-naive cells displaying the subset of genes that significantly correlate with WNT5A expression. The top 5 rows represent relative expression changes in naive cell lines compared with BRAFi-resistant (-R) cell lines generated by chronic treatment with PLX4032. The lower 5 rows display the relative expression of these genes in naive melanoma cells in comparison with a reference data set determined as an average of the expression profiles of 47 different melanoma cell lines (Mel-RefMixB).

Figure 2. WNT5A loss of function reduces melanoma cell number.

(A) Growth curves of A375 (left) and MEL624 (right) melanoma cells transfected with control siRNA #1, WNT5A siRNAs #1 and #2, and BRAF siRNA as a positive control. Error bars indicate SD calculated from 3 independent experiments with triplicate wells counted for each time point. (B) Average number of colonies per 100 A375 (left) or MEL624 (right) melanoma cells plated in soft agar, scored 1 week after siRNA transfection and embedding in soft agar. Error bars indicate SD calculated from 3 independent experiments with duplicate wells for each siRNA. (C) Representative photographs of A375 cells grown in 3D Collagen I culture after siRNA transfection. (D) Quantification of A375 spheroid area at day 4 following siRNA transfection. Each point represents the area of a single melanosphere determined by image analysis. Center bars indicates the mean melanosphere area, and error bars show the SD. See Methods for further details on measurement procedure. Statistically significant differences in colony formation in soft agar and in melanosphere size were calculated using 1-way ANOVA. (**P < 0.01, ***P < 0.001.)

Figure 3. WNT5A loss of function decreases the viability of both PLX-naive and PLX-resistant melanoma cells.

(A) Quantification of the percentage of A375 cells that were apoptotic (TUNEL⁺) after siRNA transfection and treatment with either DMSO or 2 μM PLX4720 for 2 days. Error bars indicate the SD. (*P < 0.05, 2-tailed t test.) (B) Western blot analysis of lysates from A375 (lanes 1–6) or MEL624 cells (lanes 7–12) transfected with the indicated siRNAs and then treated 2 days later with either DMSO (lanes 1–3, 7–9) or 2 μM PLX4720 (lanes 4–6, 10–12) for 48 hours. Blots were probed with either PARP antibody or β-tubulin antibody as a loading control. (C) Normalized viability of A375 cells after transfection with siRNAs followed by treatment with increasing doses of PLX4720 for 2 days. Normalized viability was determined with the mock + DMSO condition set at 100%. Error bars show SD calculated from 3 independent experiments. Nonlinear best fit curves were obtained using GraphPad software (control siRNA #1, R² = 0.9472; control siRNA #2, R² = 0.9144; and WNT5A siRNA pool, R² = 0.9016). (D) Normalized viability of A375-R (left) and MEL624-R (right) cells that were plated in medium containing 2 μM PLX4720 48 hours after siRNA transfection and allowed to grow for 3 days. Data were normalized to the control siRNA #1 condition set to 100%, and error bars indicate SD of 3 independent experiments. (**P < 0.01, 2-tailed t test.)

Figure 4. WNT5A overexpression enhances melanoma cell proliferation.

(A) Representative Western blots of lysates collected from A2058 and A375 stable cell lines generated by infection with either WNT5A-IRES-GFP or IRES-GFP control lentiviruses. Blots were probed with antibodies to detect the expression of WNT5A and total ERK1/2. (B) Representative photographs of colonies formed 3 weeks after embedding of 100 A375 cells or 1,000 A2058 cells in soft agar. (C) Average number of colonies formed in anchorage-independent growth conditions per 100 A375 cells (left) or 1,000 A2058 cells (right) plated in soft agar. (D) Quantification of A2058 soft agar colony area. Each point represents the area of a single colony determined by image analysis. Center bars indicate the mean colony area, and error bars show the SD. Statistical significance of differences in colony number and melanosphere size was calculated using a 2-tailed Student’s t test. (**P < 0.01, ***P < 0.001.) (E) Summary of the spheroid-forming potential of SK-MEL-28 cells infected with either WNT5A or GFP control lentiviral particles. Representative photographs of melanospheres taken 10 days after plating of single cells are included below the table summarizing the proportion of sphere-forming cells compiled from 3 independent experiments. (F) Kaplan-Meier survival curve indicating the tumor-free survival of mice injected with 50,000 A2058 melanoma cells expressing either WNT5A or GFP controls.

Figure 5. WNT5A enhances AKT pathway activity in melanoma.

(A and B) Cell lysates analyzed by Western blotting. Blots were probed with antibodies to detect total ERK, AKT, and HSP90 proteins as well as the phosphorylated forms of ERK1/2 (Thr202, Tyr204), AKT (Ser473), and PKC (pan-phospho-βII Ser660). In A, melanoma cell lysates were collected after transfection of A375 cells with control siRNA #1 (lanes 1–3) or WNT5A siRNAs (lanes 4–12) followed by stimulation with 5% FBS for 0, 10, or 30 minutes. In B, protein lysates were collected from either naive A375 cells (lanes 1–3), A375-R cells (lanes 4–6), naive MEL624 cells (lanes 7–9), or MEL624-R cells (lanes 10–12) that were treated with either medium, DMSO, or 2 μM PLX4720 for 24 hours before sample collection.

Figure 6. The WNT5A receptors FZD7 and RYK promote melanoma cell growth and AKT phosphorylation.

(A) Average number of colonies formed in soft agar after transfection of A375 (left) and MEL624 (right) cells with pooled siRNAs followed by culture in soft agar for 1 week. (B) Quantification of A375 melanosphere area. Statistical significance of differences in soft agar colony number and in melanosphere size was calculated by ANOVA. (**P < 0.01, ***P < 0.001.) (C) Normalized viability of A375 cells that were transfected with the indicated siRNAs, then subsequently treated with increasing concentrations of PLX4720 2 days later. Transfected cells were grown in the presence of PLX4720 for a total of 3 days before viability was determined. Values were normalized to control siRNA with DMSO set at 100%, and nonlinear, best fit regression curves were generated using GraphPad. (D) Average cell viability of A375 cells plated at a density of 5,000 cells per well in 96-well plates 2 days after transfection with either control siRNAs or siRNAs targeting WNT5A, RYK, and FZD7. Cells were then allowed to grow for 3 days, and cell viability was determined. All siRNAs are P < 0.0001 by 1-way ANOVA and Dunnett’s post-test comparison with control columns. (E) Western blots of lysates from A375 cells transfected with either control siRNA #1 (lanes 1–3), pooled FZD7 siRNAs (lanes 4–6), or pooled RYK siRNAs (lanes 7–9). Blots were probed with antibodies to detect the phosphorylated forms of ERK1/2 (Thr202, Tyr204), AKT (Ser473), and PKC (pan-phospho-βII Ser660) and HSP90 as a loading control.

Figure 7. WNT5A is upregulated as part of a gene signature associated with clinical phenotypes in melanoma.

(A) The gene set upregulated in conjunction with WNT5A in BRAFi-resistant melanoma cells from Figure 1D was assessed in the previously published Mannheim data set, in which melanoma samples were clustered on the basis of either a highly metastatic phenotype (cohort C) or a less metastatic phenotype (cohorts A and B). The gene probes in the data set that were significant by ANOVA analysis (P < 0.01) are displayed in this heat map. Signal intensities are shown as log₂-transformed data. (B) The same gene set from Figure 1D was assessed in the Broad Institute’s database of 88 melanoma samples (cell lines and short-term cultures). The heat map shows probes from the gene set that are significantly different (t test, P < 0.01) between samples with low and high expression of WNT5A.