Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [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 7080.