Purpose: Melanoma cells are resistant to endoplasmic reticulum (ER) stress-induced apoptosis. Ubiquitin proteasome pathway is vital for cells to restore normal ER function. Ubiquitin-conjugating enzyme E2C (UBE2C) participates in cell cycle progression and checkpoint control by degradation of mitotic cyclins. We examined UBE2C's biological function in melanoma cell growth.
Experimental Design: Tissues from patients with primary and metastatic melanoma were collected. BRAF and NRAS mutation status were determined. We examined the gene expression profiles using cDNA microarray. 8261 differentially expressed genes were identified between primary and metastatic melanoma and were submitted to Panther classification system analysis for cluster analysis. The genes involved in the ubiquitin proteasome pathway were of most significant interest due to its role in the mediation of p53 degradation and ER stress mechanism. RNA was extracted from primary and metastatic melanoma tissue samples where cDNA was synthesized. Expression levels of UBE2C gene were determined based on the levels of the target message relative to the house keeping gene, GAPDH. Gene silencing was performed using UBE2C siRNA method. Growth inhibition was examined by treating melanoma cell lines with UBE2C siRNA and subsequently treating with PLX4032 at various concentrations.
Results: The microarray analysis showed the basal level of UBE2C mRNA was highly expressed in several melanoma cell lines. UBE2C gene expression was higher in metastatic melanoma than that in primary melanoma of any tumor thickness ((1.23±0.14 vs 0.64±0.07, p<0.01). Microarray analysis from human melanoma cell lines showed that UBE2C basal expression was high cross the melanoma panel and the expression level was different among melanoma cell lines. UBE2C gene silencing significantly inhibited melanoma cell growth at day 4 (73.8%) and day 6 (83.8%) after UBE2C siRNA treatment. Cell death study demonstrated that the percent of apoptosis in cell lines treated with UBE2C siRNA_8 (22.1±0.89 vs 9.2±1.7, p<0.05) was significantly higher than cells treated with siRNA control oligos. Furthermore, BRAF inhibitor, PLX4032, decreased UBE2C expression through the MAPK pathway inhibition. UBE2C gene silencing sensitized melanoma cells to BRAF inhibitor and resulted in a higher level of apoptotic cells and cell cycle G2-phase arrest. The combination of PLX4032 and UBE2C silence has an additive effect on growth inhibition. Combination of UBE2C gene silencing and PLX4032 significantly inhibited cell growth (74.8±1.1% inhibition). Microarray data showed that UBE2C mRNA level was less down-regulated in BRAF inhibitor- resistant melanoma cell lines than that in BRAF inhibitor- sensitive melanoma cell lines (0.756±0.073 vs 0.393±0.138, p<0.05).
Conclusions: Our data suggests that UBE2C may be a valuable target for primary and metastatic melanoma and those resistant to BRAF blockage associated with the MAPK pathway. The combination of a BRAF inhibitor and UBE2C silencing has a significant additive effect on the inhibition of melanoma cell growth. UBE2C gene silencing also sensitized the melanoma cells to BRAF inhibitor. UBE2C may be a powerful therapeutic target for melanoma therapy.
Citation Format: Jenny Jimmy Hong, Kewei Gong, David Kaufman, Hsiaowang Chen, Richard Essner. Ubiquitin-conjugating enzyme E2C: a potential therapeutic target for primary and metastatic melanoma by microarray gene expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B026.
- ©2016 American Association for Cancer Research.