Jinhua Wang, Sharon K. Huang, Diego M. Marzese, Sandy C. Hsu, Neal P

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Epigenetic Changes of EGFR Have an Important Role in BRAF Inhibitor–Resistant Cutaneous Melanomas Jinhua Wang, Sharon K. Huang, Diego M. Marzese, Sandy C. Hsu, Neal P. Kawas, Kelly K. Chong, Georgina V. Long, Alexander M. Menzies, Richard A. Scolyer, Sivan Izraely, Orit Sagi-Assif, Isaac P. Witz, Dave S.B. Hoon Journal of Investigative Dermatology Volume 135, Issue 2, Pages 532-541 (February 2015) DOI: 10.1038/jid.2014.418 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Epithelial to mesenchymal transition (EMT) of BRAF inhibitor (BRAFi)-resistant cells. Proliferation, migration, and invasion of BRAFi-resistant cells. (a) BRAFi-resistant melanoma cells (M14R and M219R) were more elongated and fibroblast-like in shape compared with respective parental cells (M14 and M219). Bar =100 μm. (b) Immunoblots of EMT protein markers (vimentin, fibronectin, N-cadherin, and alpha-smooth actin (SMA)). (c) Migration of melanoma parental cells (M14 and M219) and BRAFi-resistant cells (M14R and M219R). Bar=100 μm. (d) Invasion of parental cells and BRAFi-resistant cells. Bar=100 μm. (e) Cell growth in soft agar. (f) Cell growth in 3D matrigel. Migration and invasion were higher in BRAFi-resistant cells than in respective parental cells, and BRAF-resistant cells grew faster compared with parental cells in soft agar and 3D matrigel. Bar=100 μm. Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 EGFR expression in metastatic melanoma cells (panels a–c) and metastatic melanoma tissues (panels d–f). (a) EGFR mRNA expression in parental (M14 and M219) and BRAF inhibitor (BRAFi)-resistant (M14R and M219R) cells assessed by gene expression microarray (left) and quantitative real-time reverse-transcriptase–PCR (right) analyses. (b) Immunoblot of EGFR expression in cells. (c) Flow cytometric analysis of EGFR expression in cells. (d) Representative immunohistochemistry (IHC) stain of EGFR in a metastatic tumor specimen obtained before (A) and after (B) treatment with BRAFi. In picture b, melanoma has higher EGFR expression compared with normal cells in tissue. Bar=100 μm. (e) Intensity of EGFR IHC staining in melanoma tumors obtained from patients before and after treatment with BRAFi (three pre-treatment patients were not available). (f) EGFR methylation array (left) and EGFR methylation-specific PCR (right). Error bars=SD (*P <0.05). Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hypomethylation of EGFR enhancers in BRAF inhibitor (BRAFi)-resistant melanoma cells. (a) The 245 kb region surrounding EGFR gene (65 kb upstream and 180 kb downstream to the transcription start site (TSS). (b) Profiles of DNA methylation at region surrounding EGFR gene. One enhancer region located upstream of the TSS is represented by the probe #4, and the other located downstream of the TSS, specifically in the first intron of the gene, represented by probe #51 defined in HumanMethylation450K BeadChip array (Illumina), respectively. (c) There was significant correlation between DNA methylation, and expression levels were located in enhancer elements. (d) Immunoblot of EGFR expression in M14 and M219 treated with 5-azacytidine (5-Aza). EGFR expression was increased in M14 and M219 treated with 5-Aza compared with treatment by DMSO control for 72 h. Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 EGFR confers resistance to BRAF inhibitor (BRAFi) in melanoma lines. (a) Representative FACS scatter plots of treated cells stained with Annexin V (y-axis) versus propidium iodide (PI; x-axis). (b) Immunoblots of EGFR expression in M219 (left) and M14R (right) cells, before and after transfection with EGFR expression plasmid or EGFR small interfering RNA (siRNA), respectively. (c) Fluorescence microscopy of cells treated by SB590885 and stained with PI (5 μg ml–1) and SYTO-13 (1 μmol ml–1). Bar=100 μm. (d) Representative FACS scatter plots of SB590885-treated cells that were stained with Annexin V (y-axis) versus PI (x-axis). (e) The Caspase3/7-Glo luminescent assay of cells treated with SB590885. Error bars, SD (*P<0.05). Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 EGFR/PI3K/AKT pathway activation in BRAF inhibitor (BRAFi)-resistant melanoma cells. (a) Growth curve of M14 and M14R cells in RPMI-1640 medium with or without EGF. (b) The Human Phospho-RTK array assay of M14 cells (top figure) and M14R cells (bottom figure) (B1, B2 (red circle): p-EGFR; C5, C6 (blue circle):p-HGFR; D17, D18 (green circle): p-EphR). Both pictures were exposured for 10 seconds. (c) Immunoblots of p-EGFR (Tyr1068) expression in parental and resistant cells. (d) Expression of EGFR in three BRAF V600 mutation (BRAFmt)-bearing cell lines: M14, M219, and M225. (e) Immunoblots show that MAPK/ERK kinase (MEK) inhibitor AZD6244 blocked MEK-mediated regulation of EGFR in BRAFmt cells. (f) Immunoblots of AKT and phosphorylated-AKT expression in M14 and M14R cells exposed to EGF after a 1-hour treatment with AKT inhibitor. (g) Immunoblots show expression of components on the PI3K/AKT pathway. Expression levels of p110 alpha, phosphorylated-p85 (Tyr458), p-AKT (473), and phosphorylated 4E-BP1 were higher in resistant cells than in parental cells. Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Response of cells exposed to EGFR inhibitor (EGFRi) and BRAF inhibitor (BRAFi) in a 3D-tumor-like microenvironment. (a) M14 parental cells and M14R resistant cells grown in a 3D-tumor-like microenvironment were treated with DMSO, BRAFi (SB590885), and/or EGFRi (erlotinib). Bar=50 μm. (b) Flow diagram of EGFR/PI3K/AKT pathway activation in resistant cell lines treated by BRAFi. The EGFR/PI3K/AKT pathway was activated when the RAS/RAF/MEK/ERK pathway was blocked by BRAFi. Journal of Investigative Dermatology 2015 135, 532-541DOI: (10.1038/jid.2014.418) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions