Combination Treatments to Overcome Fibroblast-Associated Resistance to BRAF Inhibitors in Malignant Melanoma Use of the PeggySue Technology to Explore Drug Responses at a Protein level

Malignant melanomas are one of the most devastating forms of cancers with high mortality rate. The melanoma cells readily spread to distant organs, where the cancer cells interact with stromal cells. Such interaction can induce protection against treatment practiced in the clinics. This project has verified a protection against the BRAF inhibitor (BRAFi) Vemurafenib when metastatic melanoma cells were grown as co-cultures together with the stroma cells, fibroblasts. Previously, it has been proposed by our group that the BRAFi resistance in the co-cultures can be associated with the PI3K-pathway. By combining the BRAFi with the PI3K inhibitor buparlisib, an enhanced anti-cancer effect in the co-cultures was observed. To investigate molecular responses to buparlisib in our in vitro-systems, we chose to utilize a previously unused method called PeggySue Charge, and attempted to detect AKT levels as an indicator of PI3K-pathway activity. However, further optimization is needed to get reliable results on AKT, or other phospho-proteins, by this method. Thus, verification of PI3K-pathway involvement in stroma-dependent BRAFi resistance at the molecular level is still lacking. Phenotypic-switching is a common characteristic of metastatic melanomas, and the mesenchymal phenotype has been associated with treatment-resistance. With a new method called PeggySue Size, we have managed to detect increased levels of mesenchymal phenotype-related proteins in melanoma cells cultured with fibroblasts. Inhibition of a WNT-pathway’s negative regulator GSK-3β, by the drug AR-A014418, induced a strong anti-cancer effect in fibroblast associated melanoma cells. However, the molecular mechanism behind this effect has not been disclosed. In conclusion, this work addressed both, a biological question on stroma-mediated resistance to BRAFi, and a technical question, how to analyze proteins with a new technology PeggySue. When optimized, the latter can contribute as a convenient tool to explore mechanisms involved in stroma-mediated resistance.