Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3
Ethan V. Abel, Kevin J. Basile, Curtis H. Kugel III, Agnieszka K. Witkiewicz, Kaitlyn Le, Ravi K. Amaravadi, Giorgos C. Karakousis, Xiaowei Xu, Wei Xu, Lynn M. Schuchter, Jason B. Lee, Adam Ertel, Paolo Fortina, Andrew E. Aplin
Ethan V. Abel, Kevin J. Basile, Curtis H. Kugel III, Agnieszka K. Witkiewicz, Kaitlyn Le, Ravi K. Amaravadi, Giorgos C. Karakousis, Xiaowei Xu, Wei Xu, Lynn M. Schuchter, Jason B. Lee, Adam Ertel, Paolo Fortina, Andrew E. Aplin
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Research Article Oncology

Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3

Abstract

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

Authors

Ethan V. Abel, Kevin J. Basile, Curtis H. Kugel III, Agnieszka K. Witkiewicz, Kaitlyn Le, Ravi K. Amaravadi, Giorgos C. Karakousis, Xiaowei Xu, Wei Xu, Lynn M. Schuchter, Jason B. Lee, Adam Ertel, Paolo Fortina, Andrew E. Aplin

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Figure 2

ERBB3 is a direct transcriptional target of FOXD3.

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ERBB3 is a direct transcriptional target of FOXD3. (A) Map of the ERBB3 ...
(A) Map of the ERBB3 locus showing read coverage for IP and input; aligned reads were visualized using the Integrated Genomics Viewer 2.0 (57). Relative signal of merged ChIP experiments is represented by red peaks, while the signal of the pooled inputs is represented with light gray peaks. The intron 1 enhancer region is underlined. (B and C) WM115TR/FOXD3-V5 cells were treated with 100 ng/ml Dox (+ Dox) or without (– Dox) for 24 hours. Cells were lysed, DNA was sheared, and protein/chromatin complexes were IP with normal IgG (B and C), anti-V5 antibody (B), or anti-RNA pol II pSer2 (C). Enrichment of ERBB3 intron 1 was validated by qPCR. Enrichment of the β-actin promoter is included as a control for specificity. Results represent the mean ± SEM (n = 4). P values are indicated. (D) WM115TR/FOXD3-V5 cells were treated with or without Dox for 24 hours. qRT-PCR was performed following RNA extraction. Fold change in ERBB3 transcript was normalized to housekeeping gene EEF1A1. Results represent mean ± SEM (n = 3). P value is indicated. (E) WM115TR/FOXD3-V5 cells were treated with or without Dox (100 ng/ml) for 24, 48, or 72 hours, and then lysed and immunoblotted as indicated. (F) Lysates from WM793TR, 1205LuTR, SK-MEL-28TR, and A375TR expressing FOXD3 for 24 hours were blotted as indicated.