Abstract 4329: Targetable nodes in fibroblast-supported melanoma cells that show resistance to BRAF inhibitors
Authors: Seip Kotryna , Haselager Marco V. , Jørgensen Kjetil , Albrecht Marco , Haugen Mads H. , Egeland Eivind Valen , Lucarelli Philippe , Sauter Thomas , Engebraaten Olav , Mælandsmo Gunhild M. , Prasmickaite Lina 
Published: July 2017

Abstract
Metastatic melanoma is notorious for the ability to change its phenotype in response to signals from the microenvironment, which might influence how melanoma responds to therapy. We have disclosed an association between fibroblast-induced phenotypic alterations in melanoma and resistance to the mutated BRAF inhibitor vemurafenib (BRAFi). This signifies the need to find other targets than BRAF to eliminate stroma-influenced melanoma cells. To approach this challenge, we performed proteomic analysis and cancer drug sensitivity screening, comparing fibroblast-supported versus non-supported melanoma cells. We showed that the effect of fibroblasts was critically dependent on cell-cell proximity, where melanoma cells get trapped in a fibronectin network, produced by adjacent fibroblasts. In such environment, melanoma cells down-regulate melanocytic programs (MITF-driven), gain mesenchymal features (AXL, PDGFR, fibronectin) and activate stress/inflammatory-response signaling pathways (JNK and STAT3). Altogether, this indicates fibroblast-induced melanoma transition to a de-differentiated, mesenchymal-like, pro-inflammatory phenotype. Melanoma cells with such phenotype were less responsive to BRAF/MAPK inhibitors and a number of other targeted drugs. However, they showed enhanced sensitivity to PI3K/mTOR inhibitors and, particularly, an inhibitor of GSK3b, stimulating Wnt/b-catenin signaling. Further, we employed flow cytometry to measure the levels of Ki67 and pS6 in single melanoma cells upon different conditions/treatments. Such analysis allowed discrimination of cell subpopulations representing a proliferative and a quiescent cellular state, and nicely reflected the influence of the tested drugs in the presence or absence of fibroblasts. We observed a subpopulation of proliferative pS6high/Ki67high melanoma cells, which remained after treatment with BRAFi if fibroblasts were present. This, fibroblast-protected BRAFi-resistant cell subpopulation, could be reduced/eliminated by PI3K or GSK3b inhibitors, verifying PI3K/GSK3 as potential targets in fibroblast-rich tumors. Currently, we are using mass cytometry (CyTOF) to further characterize cell subpopulations with respect to multiple markers related to cell signaling and immune interactions. Preliminary results indicate that not only signaling protein levels, but also levels of immunoregulatory proteins are altered in melanoma cells that get support from the fibroblasts. In conclusion, we demonstrate fibroblast-induced melanoma switching to a mesenchymal-like pro-inflammatory phenotype, which favors melanoma resistance to BRAF inhibitors, but sensitizes to inhibitors of PI3K/mTOR-associated signaling. CyTOF-analysis of complex tumor-stroma cell systems is used to search for additional strategies to target stroma-supported melanoma cells, either at the level of signaling, or immune interactions.
Citation Format: Kotryna Seip, Marco V. Haselager, Kjetil Jørgensen, Marco Albrecht, Mads H. Haugen, Eivind Valen Egeland, Philippe Lucarelli, Thomas Sauter, Olav Engebraaten, Gunhild M. Mælandsmo, Lina Prasmickaite. Targetable nodes in fibroblast-supported melanoma cells that show resistance to BRAF inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2017-4329