WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors
Jamie N. Anastas, Rima M. Kulikauskas, Tigist Tamir, Helen Rizos, Georgina V. Long, Erika M. von Euw, Pei-Tzu Yang, Hsiao-Wang Chen, Lauren Haydu, Rachel A. Toroni, Olivia M. Lucero, Andy J. Chien, Randall T. Moon
Jamie N. Anastas, Rima M. Kulikauskas, Tigist Tamir, Helen Rizos, Georgina V. Long, Erika M. von Euw, Pei-Tzu Yang, Hsiao-Wang Chen, Lauren Haydu, Rachel A. Toroni, Olivia M. Lucero, Andy J. Chien, Randall T. Moon
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Research Article Oncology

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 (BRAFV600E/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.

Authors

Jamie N. Anastas, Rima M. Kulikauskas, Tigist Tamir, Helen Rizos, Georgina V. Long, Erika M. von Euw, Pei-Tzu Yang, Hsiao-Wang Chen, Lauren Haydu, Rachel A. Toroni, Olivia M. Lucero, Andy J. Chien, Randall T. Moon

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Figure 6

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

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The WNT5A receptors FZD7 and RYK promote melanoma cell growth and AKT ph...
(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.