Targeting MAPK Signaling in Melanoma Cells : Implications for Immune Recognition and Cell Fate

Abstract

To date, there are no encouraging treatment options for patients with malignant melanoma available. In search of therapeutic opportunities for these patients, the MAPK pathway called attention due to the discovery that components of this pathway are frequently mutated in cutaneous melanoma cells. 44% of melanoma patients have activating mutations in BRaf, and a single mutation, V600EBRaf, accounts for 90% of these BRaf mutations. Upstream, NRas is mutated in 22% of patients. The two mutations are mutually exclusive, indicating that one “driver” mutation is sufficient to constitutively activate the pathway. Several small molecule inhibitors have been developed to inhibit MAPK signaling by targeting BRaf or MEK. In a system comprising a V600EBRaf mutated melanoma cell line and three primary melanoma lines from one patient which carry a G469RBRaf mutation, I have comprehensively analyzed the effects of MAPK inhibition through U0126 and AZD6244 (specific MEK inhibitors) and Sorafenib (multi-kinase BRaf inhibitor) on recognition by NK cells, modulation of other cell signaling pathways in melanoma cells and their fate between apoptosis and autophagy. Surface expression of NK cell ligands on melanoma cells was strongly modulated by MAPK inhibition. Expression of CD155, a ligand for the activating NK cell receptor DNAM-1 was decreased following MEK inhibition with U0126 and AZD6244 but increased by Sorafenib-mediated BRaf inhibition. This regulation of CD155 in melanoma cells had functional consequences for activation of NK cells because reduced CD155 expression following MEK inhibition correlated with reduced levels of NK cell cytotoxicity. Besides the analysis of NK cell activation against treated melanoma cells, the tumor cell fate was also investigated in detail. The core of my comprehensive analysis was based on the simultaneous inspection of multiple cell signaling pathways in tight kinetics up to 96 hours. I was able to unravel differences in the mechanisms of action between the two MEK inhibitors and the BRaf inhibitor with respect to phosphorylation of MEK and kinase stability in general. In addition, I could identify consequences of MAPK inhibition on Akt, JNK, and p53 signaling pathways, which are all involved in the regulation of apoptosis and autophagy. En route to the evaluation of autophagy regulation in response to MAPK treatment, I have established a new ImageStream-based method for the quantification of autophagy, combining flow cytometry and single cell imaging. JNK was identified as a critical negative, and NFκB as a positive regulator of autophagy. The combination of this quantification of autophagy with an approach to measure apoptotic cells allowed the simultaneous detection of autophagy and apoptosis in the same cell under conditions of MAPK inhibition. These results indicate that Sorafenib, but not U0126 or AZD6244, effectively inhibits autophagy and promotes apoptosis. Taken together, my studies clearly demonstrate that interference of the oncogenic MAPK pathway has consequences for immune recognition of tumor cells, the tumor microenvironment and the balance between autophagy and apoptosis.