Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach
Authors: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump
Journal: Cancer Research (2024)

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5445.