Abstract 1899: Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma

Abstract

Abstract Melanoma is the most aggressive of all skin cancers, being the leading cause of death for cutaneous malignancy, with elevated metastatic spread and drug resistance. Because the deregulation of components of lipid metabolism may contribute to melanoma cell resistance, the aim of this study was to examine the dependence of melanoma cell growth on extracellular lipids, the associated lipid metabolism gene expression profiles and lipid impact on sensitivity to BRAF inhibitors. We applied preclinical pharmacology approaches including western blot analyses, growth inhibition assays quantitative Real-time PCR (qRT-PCR) in melanoma cell lines including variants with acquired resistance to BRAF inhibitors. Two matched pairs of sensitive and PLX4032-resistant cell lines (LM16 and LM36) were used. Both pairs carried V600E BRAF gene mutation and BRAF gene amplification, and LM36R displayed mutant NRAS. When compared to the parental sensitive cells, LM16R cells showed increased activation of MAPK-ERK and PI3K-Akt-mTOR pathways, while LM36R cells displayed increased activation of IGF1R and LKB1. Under standard culture conditions, when compared to the parental sensitive cells, both the resistant variants showed reduced expression of DHCR24 (24-Dehydrocholesterol Reductase), and LM16R showed reduced protein level of the lipogenic enzyme FASN (Fatty Acid Synthase) while LM36R cells displayed similar FASN protein levels. When grown in lipid-free medium, LM16 and LM16R cells increased the expression of both FASN and DHCR24 proteins. These changes were particularly evident for DHCR24 in LM16R cells after 48 h of growth in the absence of lipids. Lipid starvation markedly reduced cell growth of LM16-LM16R as well as LM36-LM36R pairs over time, albeit with a lower impact at higher cell density. Growth inhibition assays indicated that all the cells cultured in lipid-free medium were more sensitive to PLX4032 than those cultured in standard medium. An analysis of the expression of lipid metabolism genes by qRT-PCR showed that after 48 hours under lipid-free culture conditions the levels of FASN were slightly increased in LM16 and LM16R cells but not in the LM36 pair. Conversely, the mRNA levels of DHCR24 were enhanced upon lipid starvation only in LM16 and LM16R cell lines. Besides, under these conditions, HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase) mRNA levels increased in LM16, LM16R and LM36R cell lines. ACSS2 (Acetyl-coenzyme A synthetase 2) was increased particularly in the LM16 cells. These findings suggest that lipid starvation redirects lipid pathways toward cholesterol synthesis (acetate-cholesterol) by increasing ACSS2, HMGCR as well as DHCR24 levels. Thus, extracellular lipid availability may influence tumor cell response to treatment, a finding to be considered in the frame of a personalized therapy tailored to the specific characteristics of each patient. Citation Format: Giovanni L. Beretta, Elisabetta Vergani, Cristina Corno, Stella Tinelli, Monica Rodolfo, Laura Gatti, Paola Perego. Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1899.