The Role of Kinesin Motor Proteins in Mammary Epithelial And Breast Tumor Cell Microtentacles

Abstract

Breast 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.