Abstract 270: The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells

Abstract

Abstract Metastasis of breast cancer (BC) to bone results in severe bone loss, fractures, and death. The crosstalk between BC cells and bone resident cells dramatically increases osteoclast activity, resulting in the release of growth factors from the bone matrix that causes aggressive tumor growth and bone loss. A potentially important aspect of this process is vesicular trafficking on microtubules (MTs) which can affect the output of signaling pathways and secretory activity of metastatic bone cells. MTs are cytoskeletal filaments composed of heterodimers, α- and β-tubulin. The tubulin isotypes and their variety of post-translational modifications (PTMs) control the properties and functions of MT filaments, a concept known as the ‘tubulin code’. Recent studies show an emerging link between alterations of the tubulin code with poor prognosis of breast cancer. However, the regulation of the code in metastatic bone cells is currently unknown. Tubulin acetylation occurs via α-tubulin N-acetyl transferase-1 (αTAT1) and can be reversed by histone deacetylase-6 (HDAC6). MTs lacking acetylation lose flexural rigidity and are prone to breaks following repetitive bending during vesicular trafficking. We found that HDAC6 interaction with α-tubulin is inhibited by a Runt-related factor (Runx2). Our biochemical, mass spectrometry and IPs analyses of MTs revealed that loss of Runx2 can reduce (i) acetylation and stability of MTs, (ii) interaction of HSP90 with α-tubulin, and (iii) levels of β 2a-tubulin. Our studies with Runx2 mutants indicate that the C-terminal of Runx2 serves a scaffolding function by interacting with MTs and HDAC6. Confocal microscopy revealed reduced puncta and altered distribution of endosomal vesicles and autophagosomes with Runx2 silencing. As MT targeting agents are often used as chemotherapeutics, we found that loss of Runx2 sensitizes breast cancer cells to docetaxel and vinblastine and reduces the secretion of IL-6. We found MDA-MB-231 cells metastasized to the bone show significant differences in microtubule isotype expression, with 10 of 19 studied showing 1.3 to 4.1 fold increases. Further analysis of whether Runx2 knockdown affects isotype expression, we found the majority of the 19 studied remain unchanged. Immunohistochemistry for Ac-α-Tub levels in bone metastatic patient samples shows significantly more Ac-α-Tub positive cells in metastatic bone tumors than primary tumors. These findings suggest a novel control mechanism of MTs stability via Runx2-HDAC6 interactions that can impact trafficking and cellular activity. Our results indicate that inhibition of Runx2 may sensitize metastatic tumors to MT targeting agents, and Runx2/HDAC6/Ac-α-Tub levels may serve as markers for metastatic tumors to help stratify patients for optimal treatment for metastatic bone disease. Citation Format: Shreya Patel, Marcus Winogradzki, Ahmad Othman, Waddell Holmes, Jitesh Pratap. The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells [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 270.