WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors
Jamie N. Anastas, … , Andy J. Chien, Randall T. Moon
Jamie N. Anastas, … , Andy J. Chien, Randall T. Moon
Published May 27, 2014
Citation Information: J Clin Invest. 2014;124(7):2877-2890. https://doi.org/10.1172/JCI70156.
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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 4

WNT5A overexpression enhances melanoma cell proliferation.

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WNT5A overexpression enhances melanoma cell proliferation. (A) Represent...
(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.