P2.01-068 Analysis of Epithelial-Stromal Interactions and their Relevance to Lung Cancer

Abstract

The communication between epithelial cells and their underlying stroma is an important but poorly understood aspect of organismal biology. If aberrantly regulated, these interactions can prove to be tumorigenic. Although it has been known for years that cancer-associated fibroblasts (CAFs) promote and sustain the growth of tumors, the underlying mechanisms remain incompletely understood. Previous work in our lab has identified a novel mechanism of communication in which CAFs secrete cardiotrophin-like cytokine factor 1 (CLCF1), a cytokine that binds ciliary neurotrophic factor receptor (CNTFR) on tumor cells and promotes neoplastic growth. CNTFR is a component of the tripartite receptor complex formed by CNTFR-gp130-LIFR and is capable of activating several oncogenic signaling cascades, including JAK-STAT. Patient tumor and normal samples were collected and plated as CAFs and normal lung fibroblasts (NLFs), respectively. The effect of CNTFR knockdown was assessed by shRNA in A549 xenografts, and the CNTFR decoy receptor was evaluated using patient-derived xenograft (PDX) models. Independent TCGA analyses of CNTFR and CLCF1 expression levels in non-small cell lung cancer (NSCLC) patients revealed that increased levels of both genes correlate with poor patient outcomes. In isolated pairs of human CAFs and NLFs from the same patient, gene expression analyses consistently demonstrated a higher level of CLCF1 in CAFs. In vitro studies using three NSCLC cell lines–A549, H23, and H358–showed that CNTFR knockdown decreases proliferation whereas exogenous recombinant CLCF1 increases growth. We repeatedly observed decreased protein levels of phosphorylated STAT3 and phosphorylated ERK upon CNTFR knockdown, thus implicating two canonical oncogenic pathways: Jak-STAT and Ras-Raf-MEK-ERK. Using xenograft models, we found that CLCF1 overexpression by CAFs increases tumor growth while knockdown of CNTFR in lung tumor cells decreases overall growth. With the use of advanced protein engineering technology, we generated a high-affinity CNTFR decoy that inhibits CLCF1-CNTFR signaling and are currently testing this novel reagent to elucidate the mechanism by which CNTFR activation alters intercellular signaling to increase tumor cell growth. Through in vivo studies with cell lines and PDXs, we are also exploring the efficacy of this CNTFR decoy as a form of lung cancer therapy. In sum, these data indicate that CLCF1-CNTFR signaling is important for NSCLC tumor growth and is a viable therapeutic target.