Elucidating the role of aberrant microtubule nucleation in breast cancer metastasis

Breast 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.