Abstract 1451: <i>TPM4</i> isoform expression modulates a cancer stem cell phenotype in human colon cancer cells

Abstract Tropomyosin 4 (TPM4) regulates contractility and migration in many cell types and is important in the migration of colon epithelial cells. Several different alternatively-spliced TPM4 isoforms are predicted to exist in mammalian cells. However, TPM4.1 and TPM4.2, differing only in the inclusion of 1 (TPM4.2) or 2 (TPM4.2) unique exon(s) at the 5’ end, are the only two isoforms experimentally validated. We previously showed that TPM4 expression may be an early, premalignant molecular event that contributes to adoption of a tumorigenic state and, specifically, that overexpressed TPM4.2 blocks certain aspects of induced differentiation in colon epithelial cells and promotes a highly proliferative state. Many studies have shown that in vivo, tumors are heterogeneous and contain a very small subpopulation of cancer stem cells that not only drive growth of the tumor via robust proliferation but also are highly invasive and resistant to conventional anti-cancer treatments. Identifying molecular characteristics that promote cancer stem cell formation is therefore critical to developing effective strategies to treat and prevent colon cancer. Adherent LS123 cells (LS123 2D), representing minimally-invasive colon cancer cells at an early stage of tumor development, express both TPM4.1 and TPM4.2 transcripts, while nonadherent LS123 tumorspheres (LS123 3D), having a highly invasive, robustly proliferating phenotype similar to cancer stem cells, exclusively express TPM4.2. Therefore, we hypothesize that TPM4.2 overexpression, either by itself or in combination with TPM4.1 suppression, plays a role in transforming colon epithelial cells to a cancer stem cell phenotype. To test this hypothesis, we determined whether knockdown of TPM4.1 and/or overexpression of TPM4.2 will induce a gene expression pattern characteristic of cancer stem cells in the LS123 2D cell line. Specifically, we measured the expression of Sox2, Lgr5, epithelial cell adhesion molecule (EpCAM), activated leukocyte cell adhesion molecule (ALCAM)/CD166, aldehyde dehydrogenase I (ALDH1), and CD44 in LS123 2D cells treated with siRNA targeting TPM4.1 or TPM4.2 or in these cells in which TPM4.1 or TPM4.2 is overexpressed. Our results indicate that favoring TPM4.2 over TPM4.1 expression in LS123 cells increases expression of certain stem cell markers, particularly CD44 and to a lesser extent EPCAM and ALCAM, whereas favoring TPM4.1 over TPM4.2 expression shows the reciprocal effect on cancer stem cell marker expression. These results may identify distinct roles for expression of individual TPM4 isoforms in colon epithelial cells and may specifically elucidate whether TPM4.2 overexpression can contribute to a more aggressive tumorigenic phenotype. Ultimately, our studies may identify therapeutic strategies that target perturbations in cell contractility and migration at early stages of colon tumorigenesis. Citation Format: Nia Lowe, Giulio Ferrero, Barbara Pardini, Alessio Naccarati, Michael Papetti. TPM4 isoform expression modulates a cancer stem cell phenotype in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1451.