Abstract 3264: Downregulation of intersectin-1s in human lung cancer may contribute to tumorigenesis

Abstract

Abstract The expression of intersectin-1s (ITSN-1s), a granzyme B (GrB) substrate and one of the trisomic genes that may provide protection against most cancers in Down's syndrome, is significantly downregulated in human lung cancer (LC) cells and tissue. To date, there is no information on the cellular and molecular factors that modulate ITSN-1s expression or whether downregulation of ITSN-1s can contribute to LC. Thus, we hypothesize that loss of full-length ITSN-1s due to proteolytic cleavage by GrB during an inflammatory response and the interplay between the signaling events triggered by the two cleavage products, are part of a complex common molecular mechanism involved in the oncogenic transformation of cells. To address this hypothesis, we used cultured LC cells and tissue, as well as a range of biochemical (Western blot, ELISA, BrdU incorporation), molecular biology (plasmid DNA transfection, qPCR), morphological (immuno-histochemistry, fluorescence microscopy) approaches, soft agar colony formation and xenograft tumor formation in nude mice to evaluate whether downregulation of ITSN-1s can contribute to lung carcinogenesis. Western blot and immuno-histochemistry of LC cells and lung tissue lysates showed significant decrease in ITSN-1s protein levels, while qPCR revealed only slight decrease, if any, in ITSN-1s mRNA. In vitro studies showed that GrB cleaves ITSN-1s and generates a cleavage fragment (GrB-EHITSN) with high proliferative potential. Lung epithelial cells expressing the GrB-EHITSN show 40% increase in BrdU incorporation, selective activation of p38MAPK and downstream Elk-1 transcription factor. These findings are consistent with a p38MAPK and Elk-1 pro-proliferative signaling pathway regulated by ITSN-1s. Activation of p38 and Elk-1 was also detected by western blot using phospho-specific antibodies, in all LC cells investigated. Expression of the two GrB cleavage fragments of ITSN-1s, individually or together, in human lung epithelial cells exerts a modulatory effect on the balance p38/Erk1/2MAPK activation. Functional studies showed that ectopic expression of ITSN-1s in the LC cells restored the p38/Erk1/2MAPK balance, significantly reduced their proliferation rate as well as the formation of colonies in soft agar and xenograft tumors in nude mice. Altogether, our results suggest that downregulation of ITSN-1s can promote the oncogenic transformation of cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3264. doi:1538-7445.AM2012-3264