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Regulation of Class III Beta-Tubulin by Src-Mediated Tyrosine PhosphorylationProstate cancer (PCa) is the second most lethal cancer in men, accounting for an estimated 30,000 deaths in 2013. For diagnoses of advanced disease, the standard treatment is androgen deprivation therapy (ADT). Despite initial success of ADT, many PCa patients relapse into an incurable androgen-insensitive disease termed Castration Resistant Prostate Cancer (CRPC). Taxane compounds (such as docetaxel [DTX]) + Steroid (such as Prednisone) is a first-line treatment, but DTX resistance is common in patients with advanced CRPC. Tubulins are an integral part of the cytoskeleton, and play a pivotal role in cell signaling, migration, and division. They exist in vivo either as soluble monomers or as α-/β-tubulin heterodimers. These dimers are either recycled into monomers, or polymerized into microtubules (MTs). Microtubules serve a range of functions including structural support of the cell body, scaffolding for signaling molecules, and mitotic spindle support. β-tubulin is also the molecular target for taxane compounds. High expression levels of Class III β-tubulin (TUBB3, a primarily neural isoform of β-tubulin) correlate with taxane resistance and poor prognosis in several human cancers, including ovarian cancer (serous adenocarcinoma), breast cancer, non-small-cell lung cancer (NSCLC), and prostate cancer (PCa). It is known that c-Src (a known proto-oncogene) phosphorylates β-tubulins during both hematopoietic and neural differentiation. This has also been observed in leukemia cells. Despite recent advances, the relationship between Src-mediated tyrosine phosphorylation and microtubule modulation is still poorly understood, especially in the context of advanced solid malignancies. Herein we show that activated Src-kinase is able to phosphorylate TUBB3 at tyrosine 340 (Y340), and that tyrosine phosphorylation of βIII-tubulin at Y340 by Src-family kinases is critical in stabilization of TUBB3, and also plays a role in regulation of mitotic spindles. Given the clinical utility of TUBB3 as a biomarker of poor prognosis, characterizing cancer-specific post-translational modification (PTM) of TUBB3 in aggressive cancer cell types could lead to more specified biomarkers of patient outcome or therapeutic response. Additionally, a deeper understanding of the complex interplay between the Src family of kinases (SFKs) and microtubules could enable future researchers to design more specific and effective microtubule-targeted and/or SFK-targeted therapies.
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Molecular Alterations that Reduce Cortical Containment of Tubulin Microtentacles and their Impact on Breast Tumor MetastasisMortality 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.
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Acetylation of α-tubulin and its Role in Aggressive and Metastatic Breast CancerMetastatic breast cancer presents a therapeutic challenge, since existing treatments largely target primary tumor growth instead of metastatic spread. Because metastasis is the leading cause of breast cancer-related death, we must investigate new targets for the treatment of disseminated disease. This study describes a novel role for α-tubulin acetylation in metastatic breast cancer. We report that metastatic breast cancer cells have high α-tubulin acetylation that is maintained under suspended conditions and extends along microtentacles, tubulin-based protrusions that promote cell-cell and cell-substrate reattachment. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this post-translational modification has a significant impact on microtentacle frequency and suspended tumor cell reattachment. Reducing α-tubulin acetylation in metastatic breast tumor cell lines also significantly inhibits migration, but does not affect proliferation. Investigating the translational importance of this modification in over 140 breast cancer patients' matched primary and metastatic tumors, we find that α-tubulin acetylation is maintained and in many cases increased in nodal metastases, relative to the primary tumor. We also discover a strong correlation between α-tubulin acetylation and the aggressive basal-like breast cancer subtype in a large cohort of breast cancer patients. These data suggest α-tubulin acetylation may promote a more metastatic phenotype through its effects on reattachment and migration while serving as a marker of an aggressive breast cancer subtype.
