Abstract 3103: Elevated RAB27B expression promotes a stem-like phenotype in NSCLC cells and enhances extracellular vesicle secretion

Abstract

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs, an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis were used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in H1299, PC9, HARA and A549 NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and metastasis. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy and immunoblotting. Our analysis revealed that RAB27B is significantly upregulated in CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of NSCLC CSCs and promotes tumor cell proliferation, angiogenesis and metastasis. EVs were positive for EV-specific markers including ALIX, TSG101, CD9, and CD81, and negative for intracellular vesicle marker Calnexin. CSCs secreted significantly more EVs than BCCs which was decreased upon KD of RAB27B in CSCs. Taken together, our data show that RAB27B plays an important role in NSCLC CSC behavior and RAB27B overexpression enhances EV secretion. Future work will focus on defining the role of RAB27B-dependent EV secretion in the interaction of the NSCLC CSC niche with BCCs and the tumor microenvironment. Citation Format: Kayleah M. Meneses, Kayla Lewis, Danielle Beetler, Bella Hughes, Verline Justilien. Elevated RAB27B expression promotes a stem-like phenotype in NSCLC cells and enhances extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3103.