Abstract 965: 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment

Abstract

Abstract Melanoma cell adhesion molecule (MCAM) is a protein located on the cell surface that was first discovered in advanced primary tumors and metastatic lesions. Overexpression of MCAM is associated with triple-negative breast cancer, decreased patient survival, and induces vimentin and slug indicative of an epithelial to mesenchymal transition (EMT). Additionally, breast cancer cell lines overexpressing MCAM show an increase in migration, invasion, and tumorigenesis. Given that overexpressed MCAM causes increased migration and invasion in multiple cancer types, MCAM could be one of the major contributors to recent studies showing increased metastatic phenotypes with certain cancer treatments. Decitabine (5-Aza-dC) is a chemotherapeutic used for acute myeloid leukemias with clinical trials underway for breast cancer use. 5-Aza-dC results in the depletion of DNA methyltransferase and acts as a demethylating agent. In 2019, 5-Aza-dC was reported to increase mRNA and protein levels of MCAM in various breast cancer cell lines, followed by an increase in EMT markers. Studies suggest that 5-Aza-dC treatment in chemo-sensitive ovarian cancer cell lines results in increased EMT markers as well as increased invasion and migration. Microtubules are a very common target of multiple cancer therapies. However, the impact of 5-Aza-dC and MCAM on the microtubule network and non-adherent cells has not been well studied. We set out to explore changes in microtentacle (McTN), cell attachment and clustering, and microtubule modifications due to 5-Aza-dC treatment and MCAM expression. Treatment of MCF10A, MCF10A variants, and MCF7 breast cancer cell lines with 5-Aza-dC showed consistent increases in the expression of MCAM. When exploring the effects of 5-Aza-dC treatment on the cytoskeleton, specifically microtubules, it was found that there is a significant increase in acetyl tubulin and detyrosinated tubulin, two common microtubule modifications correlated with an increase of McTNs. However, there was not a significant increase in the number of cells with McTNs in treated cells. Since McTNs can aid in attachment and cell clustering, attachment assays and clustering assays were performed after 5-Aza-dC treatment. These experiments showed that 5-Aza-dC decreased the ability of all cell lines to attach and impeded these cells' ability to form clusters. While there are emerging concerns that Decitabine can increase metastatic phenotypes of migration and invasion in adherent cells, our results indicate that within a non-adherent environment, Decitabine reduces the metastatic phenotypes of clustering and reattachment. Ongoing experiments focus on testing the impact of stable genetic alterations in these cell lines to examine the specific impact of MCAM overexpression on the microtubule network and metastatic potential. Citation Format: David Annis, Keyata Thompson, Julia Ju, Makenzy Mull, Trevor Mathias, Michele Vitolo, Stuart Martin. 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 965.