Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies which remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this post-translational modification exerted a significant impact on microtentacle frequency and the re-attachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of over 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared to primary tumors. Proteomic analysis of an independent cohort of over 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in breast cancer patients.

Epithelial-to-mesenchymal transition (EMT) is associated with increased breast tumor metastasis, but the specific mechanisms by which EMT promotes metastasis remain somewhat unclear. Despite the importance of cytoskeletal dynamics during both EMT and metastasis, very few current studies examine the cytoskeleton of detached and circulating tumor cells. Specific post-translational α-tubulin modifications are critical for adherent cell motility and implicated in numerous pathologies, but also remain understudied in detached cells. We report here that EMT induced through ectopic expression of Twist or Snail promotes α-tubulin detyrosination and the formation of tubulin-based microtentacles in detached human mammary epithelial cells. Mechanistically, EMT downregulates tubulin tyrosine ligase enzyme resulting in an accumulation of detyrosinated α-tubulin (Glu-tubulin), and increases microtentacles that penetrate endothelial layers to facillitate tumor cell reattachment. Confocal microscopy demonstrates that microtentacles are capable of penetrating the junctions between endothelial cells. Suppression of endogenous Twist in metastatic human breast tumor cells is capable of reducing both tubulin detyrosination and microtentacles. Clinical breast tumor samples display high concordance between Glu-tubulin and Twist expression levels, emphasizing the coupling between EMT and tubulin detyrosination in vivo. Coordinated elevation of Twist and Glu-tubulin at invasive tumor fronts, particularly within ductal carcinoma in situ samples, establishes that EMT-induced tubulin detyrosination occurs at the earliest stages of tumor invasion. These data support a novel model where the EMT that occurs during tumor invasion downregulates tubulin tyrosine ligase, increasing α-tubulin detyrosination and promoting microtentacles which could enhance the reattachment of circulating tumor cells to the vascular endothelium during metastasis.

The cytoskeletal organization of detached and circulating tumor cells is currently not well-defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, circulating tumor cells reattach in distant tissues via a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of circulating tumor cells match exactly with the mechanisms supporting tubulin microtentacles, which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how microtentacle formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to microtentacles and is both necessary and sufficient to promote microtentacle extension in detached breast tumor cells. Tau-induced microtentacles increase reattachment of suspended cells and retention of circulating tumor cells in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through microtentacle formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.