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 3

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

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WNT5A loss of function decreases the viability of both PLX-naive and PLX...
(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.)