J. Sheldon1, G. Khaushik1, P. Dandawante1, S. Anant1, J. M. Mammen1 1University Of Kansas,Surgery,Kansas City, KS, USA
Introduction:
The incidence of melanoma has increased dramatically over the last few decades making it one of the fastest growing malignancies in the United States. Melanoma expresses a plastic and aggressive phenotype, lacking the majority of regulatory mechanisms due to the aberrant activation of various signaling pathways. In this context, aberrant activation of the Notch signaling pathway in melanoma has also been reported by our laboratory as well as by others. Also of importance, the oncogenic mutation predominantly at codon 600 in the BRAF gene (present in nearly 40–50% of melanoma patients) has changed treatment options for melanoma over the past decade. Unfortunately, recent studies report that the majority of patients treated with the BRAF inhibitors vemurafenib and dabrafenib eventually develop drug resistance. The mechanisms of resistance are still poorly understood. In the present study, we explore the role of Notch signaling in vemurafenib induced drug resistance in melanoma cells.
Methods:
For our experiments, we used various melanoma cell lines (SKMEL-28, UACC275 and A2058 cells). The hexoseaminidase assay was used to determine cell proliferation and to calculate the IC50 (drug concentration causing 50% growth inhibition) in drug sensitive and drug resistant cell lines. Drug-resistant cells were developed by repeatedly growing cells in culture media with increasing doses of drug with time. The surviving daughter resistant cells were compared to the parental sensitive cells using proliferation assay. The IC50 for these paired cell lines was used to determine the increase in resistance. Protein expression studies in cells were done by using standard immunoblotting techniques.
Results:
We observed an increased IC50 value of vemurafenib in drug resistant UACC275 cells. IC50 values at the 72hr time point for UACC-RV-10, UACC-RV-10(1), UACC-RV-15(1) and UACC-RV-15(2) (UACC275 isogenic cells) were ~15, 14, 25 and 22 µM respectively as compared to the IC50 value for parental wild type UACC275 cells of ~0.50 µM. We observed a similar pattern of increased IC50 values in other melanoma cell lines (B16/F10, SKMEL-28, A2058, M14, and melanoma patient derived cells). After several passage of culture in vemurafenib, we also noted changes in cell morphology with cells becoming small and round compared to their usual elongated and stretched appearance. We further evaluated the expression pattern of various Notch receptors in drug sensitive cells as compared to drug resistant isogenic cell lines. Levels of cleaved Notch-2 (10 fold) and -4 (3 fold) were significantly higher in drug resistant cells.(n=5)
Conclusion:
A reductionist model of vemurafenib resistance can be developed using the UACC275 cell line. Both Notch-2 and -4 levels are higher in vemurafenib resistant melanoma cells in this model. Notch signaling may directly or indirectly play an important role in the development of vemurafenib drug resistance in melanoma cells.