Molecular Alterations that Reduce Cortical Containment of Tubulin Microtentacles and their Impact on Breast Tumor Metastasis

Abstract

Mortality among breast cancer patients is largely a measure of the degree to which primary tumors can successfully spread throughout the body and colonize secondary organs. Furthering our understanding of the metastatic process is therefore central to the development of effective treatment strategies. The primary means of dissemination during metastasis is via entrance into the lymphatic or circulatory vasculature and subsequent exit in another part of the body. To accomplish this, cells must detach from the extracellular matrix (ECM) and neighboring cells to which they are normally anchored, and undergo indefinite periods of suspension, during which time they continue to respond to their microenvironment. We have found that this detachment induces the extension of dynamic tentacle-like protrusions of the plasma membrane called microtentacles (McTNs), which are frequent in highly metastatic tumor cells. As these structures have been implicated in a role for re-attachment to extracellular matrix, it is important to elucidate the mechanisms that drive their formation, as well as the manner in which they respond to common cancer therapies, in order to develop a thorough understanding of their contributions to breast tumor metastasis. Anti-mitotic cytotoxic agents are one of the most commonly employed chemotherapies, yet the manner in which these chemotherapeutics impact the metastatic process remains unclear. Specifically, it is important to consider how circulating tumor cells respond when exposed to these compounds. Experiments were conducted to address this question, and revealed that these microfilament and microtubule binding chemotherapeutics both enhanced McTN formation, and promoted adhesion to an ECM in a manner consistent with reports of endothelial docking by circulating tumor cells. Such findings are clinically valuable, as they suggest that these common therapies (specifically Taxol) could inadvertently enhance the survival of chemotherapy-resistant tumor cells. To better understand the mechanisms by which cytoskeleton-targeted chemotherapeutics exert their effects on McTNs, a genetic approach was taken to test the idea that differential molecular control of the actin cytoskeleton accounts for the variability in McTN frequency that accompanies changes in metastatic potential. The prototypical member of the Src family of tyrosine kinases was found to negatively regulate McTN formation in a manner that was independent of invadopodia formation and function. While both of these forms of plasma membrane protrusion are frequent in cancer cell types, these findings distinguishes them as separate structures and reflects the fact that they occur separately in anchorage-dependent and detached states. Additionally, it was found that phosphorylation of the cortactin protein, which is a prominent c-Src substrate, promotes McTN formation, even in the absence of functional c-Src. Alternatively, blocking the phosphorylation of cortactin with point mutations of selected tyrosine residues impeded McTN formation, an effect that is thought to stem from altered mechanical properties and enhanced rigidity of the cortical actin cytoskeleton. Such findings emphasize the need to understand the biology and behavior of detached and circulating tumor cells; thus subsequent experiments aimed to elucidate the molecular mechanisms underlying McTN formation, revealing specific regulatory roles for c-Src and phosphorylation of cortactin within the cell cortex. These studies also provide evidence that common antimitotic chemotherapies disrupt the normal homeostasis of the cytoskeleton, and, by doing so, can recapitulate the McTN phenotype of aggressive tumor cells. Strikingly, the cytoskeletal responses to these pharmacological agents promoted ECM-adhesion in suspended tumor cells; populations which are morphologically and physiologically analogous to circulating and bloodborne disseminated tumor cells. These findings are significant for clinical consideration, and indicate that care should be taken when administering taxanes as an adjuvant or neoadjuvant therapy.