A quantitative proteomic survey reveals mechanisms of the bi-directional signaling between lung fibroblasts and lung cancer cells

Cancer cells and fibroblasts support each other for increased aggressiveness and drugs resistance. Cancer cells educate fibroblasts to acquire some cancer associated fibroblasts (CAFs) phenotype and CAFs feedback by regulating cancer cells signal rewiring in response to tyrosine kinase inhibitors (TKIs), for their survival and proliferation. The mechanisms underlying this bi-directional interplay at a systems level are poorly understood. To get insights on this complex signaling crosstalk between lung cancer cells (LCs) and lung fibroblasts (LFs), we applied a Cell Type-specific labelling using Amino acid Precursors (CTAP) to perform quantitative mass spectrometry data analysis. CTAP allows for simultaneous metabolic labelling of co-cultured cell types leading to subsequent differential proteomic analysis. We generated three sets of proteomic data including: shotgun data for the whole cell proteomes, activity- based protein profiling (ABPP) using a desthiobiotin-ATP probe to target kinase regulation and phospho-tyrosine proteomic data. We selected PC9 lung cancer cells carrying an activating EGFR mutation and the transformed lung fibroblasts (WI-38-VA13). Their mono-cultured cells are used as reference for comparative data analysis. Among the 6319 proteins identified in shotgun data, expression levels of 1305 are changed in LFs by LCs, while 1001 are changed in LCs by LFs. ABPP data consisted of 336 verified ATP-binding sites from 201 proteins including proteins, of the 26s proteasome family associated to cell death regulation, of the ATP-binding cassette family and one hundred kinases. Phosphorylation of one fourth of the (ABPP) kinases matched hundreds of known substrates regulation in phosphoproteomic data as described by PhosphositePlus database. Preliminary analysis from shotgun data using KEGG signaling pathway database, suggest enrichment of proteins of (i) insulin signaling pathways including IGF1R/IRS1/IRS2/AKT axis and (ii) changes of Ephrin receptors associated to cell migration in LFs after co-culture with LCs. Gene set enrichment analysis identified RAS signaling in LFs after co-culture with LCs. Conversely, LFs drive expression changes of AXL receptor in LCs which correlates with phosphorylation of its tyrosine residue (Y702). AXL is associated with TKIs resistance, increased cell survival and metastatic propensity of cancer cells. Its regulation is accompanied by changes of expression (Shotgun data) and activity (ABPP) of non-traditional MAP4K3 proteins as well as regulation of tyrosine phosphorylated protein YAP-1. AXL, MAP4K3 and YAP-1 are linked to hippo signaling pathway and constitute an axis for regulation of cell survival and cell migration in LCs induced by LFs. Taken together, our data nominate new mechanisms of the bi-directional signaling between LCs and LFs, as well as potential targets for therapeutic intervention. Citation Format: MARTIAL BOUTCHUENG DJIDJOU, Jae-Young KIM, Ki-Cheol Han, Gabriela Wright, Anurima Majumder, Bin Fang, John Koomen, Lily.L Remsing Rix, Uwe Rix, Eric Haura. A quantitative proteomic survey reveals mechanisms of the bi-directional signaling between lung fibroblasts and lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5089.