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Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Background: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. Methods: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot β-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. Results: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. Conclusions: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.