PO-294 BRAFV600E/PTEn-loss status is associated with interleukin (IL)−8 expression in preclinical models of colorectal cancer (CRC)

Abstract

IntroductionMutational status in CRC is a strong predictor for overall survival; unfortunately, tumour microenvironment (TME) and tumor-stroma interactions (TSI) also increase cancer cells’ drug resistance, leading to an urgent need to better understand the molecular mechanisms of acquired tumor-resistance, which remains crucial to determine overall patient benefit.Material and methodsProduction of IL-8 and vascular endothelial growth factor (VEGF) was determined by ELISA under standardised culture conditions. Modulation of cytokine production after exposure to selective inhibitors of the MAPK and PI3K pathways was assessed, by both ELISA assay and real time-PCR. BRAF, MEK1, ERK1 and ERK2 expression were modulated using siRNAs specifically targeting these genes.Results and discussionsCRC cell lines harbouring both BRAFV600E and PTEN-loss expressed the highest levels of IL-8 and a ROC curve-based prediction algorithm based on these two mutations had 68% accuracy in predicting IL-8 production (p=0.002); on the other hand, VEGF levels inversely correlated with KRAS mutational status. IL-8 is tightly and transcriptionally controlled by activation of the MEK/ERK pathway, as the MEK inhibitor trametinib downregulated ERK Thr202/Tyr204 phosphorylation in the nuclear compartment and profoundly suppressed IL-8 production regardless of the genetic background; conversely, the selective BRAF inhibitor dabrafenib downregulated IL-8 only in BRAFV600E contexts, but upregulated its production in parallel with ERK Thr202/Tyr204 and STAT3 Ser727 phosphorylation in the nuclear compartment of BRAF-wt CRC cells. PI3K/mTOR inhibitors did not significantly modulate IL-8 production, confirming that the IL-8 release is downstream the BRAF/MEK/ERK pathway.In order to better investigate the role of MAPK pathway in IL-8 regulation, components of the MAPK-signalling pathways (BRAF, MEK, ERK1 and ERK2) were silenced by siRNA: IL-8 production was significantly downregulated after BRAF, MEK, or ERK2 silencing, regardless of the BRAF mutational status; conversely, no effects on IL-8 expression were observed after ERK1 silencing.ConclusionThese results showed that, in preclinical models of CRC, IL-8 expression is regulated by a BRAF/MEK/ERK2/STAT3 axis, and differential modulation of IL-8 after pharmacological treatments is dependent on BRAF mutational status. Such evidence allows better understanding of the molecular mechanisms of chemokine regulation, which can, in turn, contribute to the development of new targeted therapies in CRC.