Abstract 2627: Acetylation of α-tubulin contributes to microtentacle formation and re-attachment in suspended breast tumor cells.
Authors: Boggs Amanda E. , Vitolo Michele I. , Bettes Rebecca A. , Slovic Jana , Charpentier Monica S. , Martin Stuart S. 
Published: August 2014

Abstract
Circulating tumor cells (CTCs) seed distant and recurrent metastases, the leading cause of breast cancer-related death. However, few laboratories are investigating cytoskeletal properties of CTCs or are targeting CTCs directly. We have shown that detached epithelial cells produce microtubule-based protrusions called microtentacles (McTNs) that facilitate CTC re-attachment. These protrusions can be stabilized by microtubule-associated proteins or post-translational modifications of α-tubulin. Acetylation is a post-translational modification that occurs on Lysine 40 (K40) of α-tubulin by a recently identified α-tubulin acetyltransferase: ATAT1. This modification is understudied in breast cancer and has not been previously examined in detached tumor cells. It is also clinically relevant, since HDAC inhibitors and microtubule-stabilizing drugs increase acetylation of α-tubulin in vivo. We have found that α-tubulin acetylation increases with metastatic potential in a panel of breast tumor cell lines and localizes to McTN protrusions in suspended metastatic cells. In tumor cells with high endogenous tubulin acetylation, immunofluorescence showed that cells transiently transfected with a non-acetylatable K40R α-tubulin mutant had fewer acetylated microtubules compared to adjacent, non-transfected cells. Functional tests in cells stably expressing K40R demonstrated that decreased α-tubulin acetylation reduced microtentacle formation. Electrical impedance also revealed decreased tumor cell reattachment in cells stably expressing the K40R mutant, compared to wild-type controls.
In tumor cells with low endogenous α-tubulin acetylation, transient transfection of the acetyltransferase ATAT1 significantly increased acetylation of α-tubulin, compared to controls on immunoblot and compared to adjacent/non-transfected cells with immunofluorescence. Live-cell fluorescence imaging showed transient ATAT1 overexpression also significantly increased microtentacle frequency. Suspended immunofluorescence showed acetylated α-tubulin localized to McTNs in ATAT1 overexpressing cells but not in controls. ATAT1 overexpression also increased tumor cell reattachment.
Since clinical tumor imaging requires a foci of more than 5 million tumor cells, current interpretations of drug effects are limited to large changes in tumor size rather than effects on metastatic dissemination. The ability of elevated α-tubulin acetylation to increase microtentacles and promote tumor cell reattachment emphasizes the importance of understanding how α-tubulin acetylation influences CTC metastasis so therapies aimed at tumor growth do not inadvertently elevate metastatic risk. Further investigation into matched metastatic breast tumor patient samples will reveal the clinical relevance of this previously understudied modification.
Citation Format: Amanda E. Boggs, Michele I. Vitolo, Rebecca A. Bettes, Jana Slovic, Monica S. Charpentier, Stuart S. Martin. Acetylation of α-tubulin contributes to microtentacle formation and re-attachment in suspended breast tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2013-2627