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The Role of Kinesin Motor Proteins in Mammary Epithelial And Breast Tumor Cell MicrotentaclesBreast cancer is the second leading cause of cancer deaths in women in the US with nearly 90% of solid tumors arising as epithelial carcinomas. Primary tumor metastasis often leads to aggressive secondary tumors starting from micrometastases that may lie dormant years after reattachment. In recent studies, detached mammary epithelial and breast tumor cells were found to produce long, dynamic protrusions composed of detyrosinated tubulin and vimentin that promote homotypic aggregation and reattachment to surfaces. These protrusions are termed microtentacles (McTNs) to distinguish them from actin-based filopodia/invadopodia and tubulin-based cilia. It is thought that McTNs function as a cell survival response to facilitate reattachment when adhesion to extracellular matrix is lost. Our goal is to elucidate the mechanisms that regulate the formation and function of McTNs by identifying key components involved in McTNs. We focused on kinesin motors that traffic cellular membranes, vesicles, and proteins along microtubules. Our long-term goal is to understand McTN structure and function to develop effective treatments that reduce the reattachment of circulating tumor cells in distant tissues. We tested the central hypothesis: kinesin motor proteins play a key role in the formation and function of McTNs. This hypothesis is based on the following evidence. 1)Circulating tumor cells bind blood vessels via a cytoskeletal mechanism consistent with McTNs. 2)Highly metastatic tumor lines display increased McTN frequencies. 3)McTNs are composed of coordinated vimentin and detyrosinated microtubules. 4)Detyrosinated microtubules and vimentin are likely cross-linked by kinesin proteins. 5)Kinesin-1 preferentially binds and traffics on detyrosinated microtubules in vivo. Using the anesthetics/kinesin inhibitors lidocaine and tetracaine, we inhibited kinesin function within cells as observed with GFP-fusion kinesin proteins, reduced McTN frequency, and decreased attachment efficiency in an effective, nontoxic concentration. lidocaine and tetracaine destabilized vimentin filament support from -tubulin as observed by immunofluorescence. Utilization of GFPKinesin1-wildtype and GFPKinesin1-mutant overexpression systems did not increase McTN frequency but increased McTN length, tubulin stability, and attachment efficiency. Partial inhibition with Kinesin-1 siRNA reduced attachment efficiency. In conclusion, Kinesin-1 is a candidate regulator of McTN formation and function and a possible therapeutic target to reduce reattachment of circulating tumor cells.
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Elucidating the Role of Aberrant Microtubule Nucleation in Breast Cancer MetastasisBreast cancer is currently the second leading cause of cancer deaths in women in the United States. The lifetime risk for U.S. women developing invasive breast cancer is 1 in 8, and 1 in 33 will ultimately die of the disease. The primary cause of death in breast cancer patients is due to metastasis of tumor cells from the primary tumor site and initiating secondary tumor sites. Our lab has recently discovered that metastatic breast tumor cells produce tentacle-like protrusions in the membrane. These protrusions, called microtentacles, appear to promote circulating tumor cells to reattach to blood vessel walls in order to eventually initiate a secondary tumor. Microtentacles have been found to be enriched with microtubules, fibers composed of the protein tubulin, a cytoskeletal protein critical in cell structure and trafficking of internal cellular constituents. We therefore examined the role of microtubule nucleation in the formation of microtentacles and breast cancer aggressiveness. We discovered that gamma-tubulin, the major protein involved in microtubule nucleation, is delocalized in aggressive breast tumor cell lines by immunofluorescence. Ultracentrifugation studies showed that this delocalization was caused by a change in the ratio of soluble and insoluble fractions of gamma-tubulin. We confirmed that gamma-tubulin is a component of microtentacles and also localizes to specific compartments in the microtentacles. Further studies determined that this redistribution of gamma-tubulin is regulated by BRCA1. When WT BRCA1 was restored in cells expressing mutated BRCA1, gamma-tubulin localization was restored to the centrosome. Furthermore, attachment assays determined that restoration of WT BRCA1 in mutant BRCA1 cells not only re-sensitized them to the microtubule targeting drug, colchicine, but also reduced rates of attachment. Together, these studies aid in the understanding of the molecular mechanisms underlying circulating tumor cell reattachment and provide possible therapeutic targets for breast cancer metastasis.
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Breast Tumor Stem Cells Have Increased Microtentacles That Can Be Targeted Therapeutically with CurcuminCancer stem-like cells (CSC) and circulating tumor cells (CTCs) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTNs), a type of tubulin-based protrusion of the plasma cell membrane which forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the HMLE breast cell line presents increased McTNs compared to its isogenic non-stem-like subpopulation, when these subpopulations are separated by flow cytometry. This increase in McTNs was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. While exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTNs in breast CSCs, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an anti-metastatic strategy.
