Abstract 927: Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors
Authors: Seip Kotryna , Fleten Karianne Giller , Barkovskaya Anna , Nygaard Vigdis , Haugen Mads Haugen , Engesæter Birgit Øvstebø , Mælandsmo Gunhild Mari , Prasmickaite Lina 
Published: July 2016

Abstract
Phenotypic heterogeneity of cancer cells can reason diversity in therapy responses within the same tumor, which might influence the overall efficacy of the treatment. Tumor stroma is an important contributor to intratumoral heterogeneity and can play a significant modulatory role in therapy response/resistance. Through studies on biological mechanisms of stroma-promoted resistance, novel targets could be identified for combination therapies aimed to eradicate both stroma-dependent and independent counterparts of the tumor.
In this study we explore how the efficacy of the BRAF inhibitor (BRAFi) vemurafenib, a targeted agent commonly used against BRAF-mutant malignant melanoma, is modulated by stromal cells. By using multiple co-culture systems and experimental metastasis models, we showed that in the presence of lung fibroblasts, adjacent melanoma cells respond poorly to BRAFi. The protective influence of stroma was associated with stroma-induced changes in the melanoma cell phenotype, which was mapped by global gene expression and proteome analysis. We revealed that under the influence of fibroblasts, melanoma cells underwent a phenotype transition to the invasive, mesenchymal-like state characterized by down-regulation of melanocytic markers (MITF and its targets), up-regulation of receptor tyrosine kinases (RTKs)/RTK-linked signaling (like AXL or PDGFR activating down-stream PI3K) and elevation of extracellular-matrix fibronectin. We propose that these alterations allow melanoma cells to utilize alternative signaling pathways, like RTK-PI3K-mTOR instead of BRAF-driven MAPK, which reduces sensitivity to BRAFi. This scenario is further supported by the observations that: i) upon BRAFi treatment, stroma-protected melanoma maintained high levels of phospho-ribosomal protein S6 (pS6), a mTOR effector protein; ii) inhibition of mTOR or the upstream pathway PI3K together with BRAF eradicated pS6high subpopulations and enhanced the anti-proliferative effect in stroma-interacting melanoma; iii) the benefit of mTOR and BRAF co-inhibition was also seen in early-stage lung metastases in vivo.
In conclusion, our findings signify the importance of stromal cells, specifically lung fibroblasts, in regulating melanoma cell phenotype and signaling, which impairs response to BRAFi. The stroma-induced invasive phenotype determinants that facilitate RTK-PI3K-mTOR signaling (e.g. AXL, PDGFR or fibronectin-binding integrins) could represent potential targets for overcoming stroma-mediated resistance to BRAFi. Currently, we are testing BRAFi in combination with several novel inhibitors of the identified RTKs and performing a cancer drug sensitivity screen to reveal the most effective drug combinations against stroma-interacting melanoma cells.
Citation Format: Kotryna Seip, Karianne Giller Fleten, Anna Barkovskaya, Vigdis Nygaard, Mads Haugen Haugen, Birgit Øvstebø Engesæter, Gunhild Mari Mælandsmo, Lina Prasmickaite. Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors. [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 927.