Abstract C114: Cancer cell induced activation of associated fibroblasts is PDGFR-mediated

Background: Recent studies have shown that tumorigenesis is not a cell autonomous process and is actively shaped by the tumor microenvironment (TME). The effect of cancer associated fibroblasts (CAFs), a critical component of the TME, on the growth, progression and drug sensitivity of the tumor itself is well studied. However, there is limited understanding about key signaling pathways activated in CAFs by cancer cells (CCs). HYPOTHESIS: CCs and CAFs talk to each other in a bi-directional manner wherein CCs alter signaling in CAFs which in turn feeds back and affects oncogenic signaling networks and behavior of CCs. In this study, we aim to better understand this bi-directional signaling at a systems level. Methods: For this, we first characterized differential tyrosine phosphoproteomes of EGFR mutant PC9 lung cancer cells and WI-38 lung fibroblasts in co-culture using mass spectrometry-based phosphoproteomics (pY). Cell type specific proteome labeling by amino acid precursors (CTAP) was used to distinguish two proteomes in co-culture. To follow up on data from the CTAP pY experiment, fibroblasts were treated with conditioned medium (CM) collected from PC9 cells and analyzed for changes in effectors of tyrosine kinase (TK) signaling, such as phosphorylation of ERK and/or AKT. Treatment with a panel of TK inhibitors coupled with chemical proteomics and siRNA approaches were used to identify the upstream kinase responsible for these signaling changes. Migration and proliferation assays were set up in the Incucyte system to study phenotypic consequences of these cancer cell-induced signaling alterations in fibroblasts. Results: The CTAP pY data shows differential regulation of receptor tyrosine kinase (RTK) signaling by co-culture in both CCs and CAFs. We focused our attention first on using a CM system to explore which potential ligands can mediate some of the CC-induced changes in kinase signaling detected in fibroblasts from the pY experiment. We found that PC9 CM induces a transient but robust increase in AKT and ERK phosphorylation in both normal as well as patient-derived lung fibroblasts. This activation was blocked by TKIs Sunitinib, Cabozantinib and Foretinib. Chemical proteomics and subsequent siRNA experiments show that the cancer cell CM-mediated AKT activation in fibroblasts is driven by PDGFR. CM-induced PDGFR activation results in activation of fibroblasts as evidenced by its increased migration/invasion capacity that can be blocked by the PDGFR inhibitor Sunitinib. We have also developed a PDGFRB/pY100 proximity ligation assay (PLA) that would allow us to annotate active PDGFR signaling complexes in situ. Conclusion: Overall, our data shows that CCs transiently trigger PDGFR-mediated TK signaling in fibroblasts thereby activating them. We next aim to understand the effect of this activation on oncogenic signaling and behavior of CCs. A better understanding of this bi-directional crosstalk may allow identification of targeted therapies against activation of CAFs that support tumor growth. Citation Format: Anurima Majumder, Martial Boutchueng-Djidjou, Jae-Young Kim, Natalia Sumi, Uwe Rix, Eric B Haura. Cancer cell induced activation of associated fibroblasts is PDGFR-mediated [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C114. doi:10.1158/1535-7163.TARG-19-C114