SUMMARY During cell division cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule co-purifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential anti-mitotic target.
The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.
Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAFV600E melanomas.
SUMMARY Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that Astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409 and is degraded during cytokinesis. Mid2 depletion led to Astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant Astrin in Astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of Astrin on K409, which is critical for its degradation and proper cytokinesis. These results may help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.
The sterol regulatory element-binding protein (SREBP) transcription factors have become attractive targets for pharmacological inhibition in the treatment of metabolic diseases and cancer. SREBPs are critical for the production and metabolism of lipids and cholesterol, which are essential for cellular homeostasis and cell proliferation. Fatostatin was recently discovered as a specific inhibitor of SREBP cleavage-activating protein (SCAP), which is required for SREBP activation. Fatostatin possesses antitumor properties including the inhibition of cancer cell proliferation, invasion, and migration, and it arrests cancer cells in G 2 /M phase. Although Fatostatin has been viewed as an antitumor agent due to its inhibition of SREBP and its effect on lipid metabolism, we show that Fatostatin's anticancer properties can also be attributed to its inhibition of cell division. We analyzed the effect of SREBP activity inhibitors including Fatostatin, PF-429242, and Betulin on the cell cycle and determined that only Fatostatin possessed antimitotic properties. Fatostatin inhibited tubulin polymerization, arrested cells in mitosis, activated the spindle assembly checkpoint, and triggered mitotic catastrophe and reduced cell viability. Thus Fatostatin's ability to inhibit SREBP activity and cell division could prove beneficial in treating aggressive types of cancers such as glioblastomas that have elevated lipid metabolism and fast proliferation rates and often develop resistance to current anticancer therapies.
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