Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells

Abstract

Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulinbased protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated A-tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated A-tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread. [Cancer Res 2008;68(14):5678-88]

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“…Similar looking structures known as "microtentacles" have been described on breast cancer cells (51,52). Microtentacles appear on detached breast cancer cells.…”

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“…3B), corresponding to the N-terminal fragment of GFAP (i.e. lacking the tail region and a part of the rod domain), which is known to perturb the entire IF network, as described with the equivalent vimentin construct (Whipple et al, 2008). Interestingly, depletion of lamin B1 also altered nucleus positioning (supplementary material Fig.…”

Histone Deacetylases as New Therapeutic Targets in Triple-negative Breast Cancer: Progress and Promises

“…Moreover, there was observed an increase of vimentin phosphorylation levels, as well as changes in vimentin filaments. Interestingly, the increased phosphorylation of vimentin led to suppression of the formation of microtentacles based on tubulin (143), which are known to help floating cells attach to other surfaces (148).…”