SOX4 Mediates BRAF Inhibitor Resistance in Melanoma through Regulation of IGF-1R Signaling: In Vitro Study

Abstract

Abstract Objective: SOX4, a transcription factor, has been found to contribute to tumorigenesis in several cancers. This study was performed to determine whether SOX4 mediates BRAF inhibitor resistance in melanoma. Methods: Melanoma cell lines with acquired resistance to BRAF inhibitor (SK-MEL-5R, SK-MEL-28R, and A375R) were generated by adding escalating concentrations of PLX4032 into parental SK-MEL-5, SK-MEL-28, and A375 cells for >6 months. The expression of SOX4 and insulin-like growth factor 1 receptor (IGF-1R) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The downstream signaling of IGF-1R was detected by Western blotting. SOX4 and IGF-1R overexpression or knockdown was conducted by lentivirus transfection. Cell viability and apoptosis were demonstrated by MTT and flow cytometry, respectively. The binding ability of SOX4 to IGF-1R promoter was determined by chromatin immunoprecipitation quantitative PCR assay. Results: SOX4 was upregulated in BRAF inhibitor-resistant melanoma cells as compared with parental cells (SK-MEL-5 group, 1.02 vs. 6.33; SK-MEL-28 group, 1.03 vs. 3.22; A375 group, 1.00 vs. 1.86; t =°7.069, 29.26, and 5.291, respectively; all P < 0.01), and PLX4032 treatment could not alter the expression of SOX4 in resistant cells. SOX4 overexpression attenuated the response of parental cells to PLX4032 (for cell viability, SK-MEL-5 group: 77.76% vs. 104.28%, F = 91.50; SK-MEL-28 group: 60.59% vs. 93.13%, F = 171.8; A375 group: 62.50% vs. 80.87%, F = 47.15. For apoptosis rates, SK-MEL-5 group: 34.90% vs. 14.31%, F = 4.781; SK-MEL-28 group, 40.8% vs. 29.4%, F = 13.32, P = 0.063; A375 group: 40.20% vs. 17.09%, F = 11.39; all P < 0.05, otherwise indicated). While SOX4 knockdown enhanced the response of resistant cells to PLX4032 (for cell viability, SK-MEL-5R group: 93.75% vs. 69.53%, F = 94.45, SK-MEL-28R group: 95.60% vs. 66.79%, F = 30.41, A375R group: 95.51% vs. 59.98%, F = 111.6; for apoptosis rates, SK-MEL-5R group: 16.2% vs. 44.4%, F = 25.67, SK-MEL-28R group: 26.59% vs. 44.20%, F = 158.0, A375R group: 5.98% vs. 31.51%, F = 14.35, and all P < 0.01). Chromatin immunoprecipitation quantitative PCR assay demonstrated that SOX4 binded to the promoter of IGF-1R (1.04 vs. 1.94 [−1044 to −920 bp] and 0.110 vs. 0.139 [GAPDH], F = 534.5, P < 0.01). In addition, SOX4 overexpression increased IGF-1R and its downstream phosphorylated ERK, phosphorylated AKT, and phosphorylated STAT3 expression, while SOX4 knockdown exerted the opposite effects. Moreover, IGF-1R knockdown overcame SOX4 overexpression-induced PLX4032 resistance (cell viability: 35.85% vs. 52.79% vs. 37.84% [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; apoptosis rates: 25.30% vs. 9.56% vs. 22.26 [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; F = 13.01 and 41.87, respectively; all P < 0.01), while IGF-1R overexpression abrogated SOX4 knockdown-induced response enhancement to PLX4032 for comparison of negative control group, sh-SOX4 group and sh-SOX4 + IGF-1R overexpressing group (cell viability: 96.62% vs. 86.86% vs. 97.26% (A375R), 98.15% vs. 81.63% vs. 98.49% [SK-MEL-5R]; apoptosis rates: 13.81% vs. 32.00% vs. 12.16 [A375R], 29.70% vs. 41.40% vs. 26.10% [SK-MEL-5R]; F = 13.56, 12.86, 38.81, and 39.85, respectively; all P < 0.01). Conclusion: SOX4 mediates BRAF inhibitor resistance in melanoma through regulation of IGF-1R signaling. SOX4 might serve as a potential target for the treatment of BRAF inhibitor-resistant melanoma.