Abstract 2953: Notch signaling is a key pathway involved in drug resistance in melanoma cells
Authors: Kaushik Gaurav , Sheldon Jonathan , Dandawate Prasad , Subramaniam Dharmalingam , Standing David , Anant Shrikant , Mammen. Joshua M.V. 
Published: July 2016

Background: Melanoma expresses a plastic and aggressive phenotype, lacking the majority of regulatory mechanisms due to the aberrant activation of various signaling pathways including the Notch pathway. Oncogenic BRAF mutation has the target for therapeutic interventions such as the drug vemurafinib but recent studies have indicated development of resistance. Unfortunately, however, the mechanisms of vemurafenib-induced resistance in melanoma are still poorly understood. We explored the role of Notch signaling in development of vemurafenib drug resistance in melanoma cells.

Methods: We have utilized melanoma cell lines (especially B16/F10, SKMEL-28, A2058, UACC275 SKMEL103 and M14) with and without the common hot spot BRAFV600E mutation for the studies. We performed hexoseaminidase and clonogenicity assays to determine the cell growth rate and IC50 values in the cell lines. To generate drug resistant cells, UACC275 and SKMEL-28 cells were repetitively grown in the presence of vemurafenib. For stem cells, we did melanosphere formation assay. For protein expression, we performed western blots.

Results: In the initial screening with vemurafinib, we observed a pattern of increased IC50 values in drug resistant cell lines, with UACC275 and SKMEL-28 being the more sensitive cells. Following sequential exposure, we developed vemurafinib-resistant cell lines, and observed that the IC50 values for proliferation inhibition to be ∼8-10 fold higher than the parental cells. Colony forming potential of the drug resistant cells was also not affected by increasing concentrations of vemurafenib, confirming acquisition of resistance. Furthermore, the drug resistant UACC275 cells presented a smaller and round morphology compared to the usual elongated and stretched appearance of the parent lines. Additionally, we also observed a significant increase in size of melanospheres for the drug resistance cells suggesting enrichment of stem cells. We further studied the expression and activation of various notch signaling cascade proteins and observed a significant increase in the levels of cleaved Notch-2, and -4 in the drug resistant cells. Interestingly, early passages of cell culture for drug resistant cells showed decrease in cleaved Notch-2 levels in cells with significant increase in basal levels of cleaved Notch-2 levels at &lt;10 passages. Therefore, a reductionist model of vemurafenib resistance can be developed using the UACC275 and SKMEL-28 cell line.

Conclusion: As Notch-2 and -4 levels were higher in most of the resistant cells, therefore, notch signaling may play critical role in the development of vemurafenib drug resistance in melanoma cells. Targeting specific notch in patients on chemotherapy especially on BRAF inhibitors will be a key event for better progression and treatment of melanoma.

Citation Format: Gaurav Kaushik, Jonathan Sheldon, Prasad Dandawate, Dharmalingam Subramaniam, David Standing, Shrikant Anant, Joshua M.V. Mammen. Notch signaling is a key pathway involved in drug resistance in melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2953.