Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.

Paclitaxel cytotoxicity in breast tumors depends on multipolar spindle maintenance and preexisting chromosomal instability.

Abstract. Hook decoration with pig brain tubulin was used to assess the polarity of microtubules which mainly have 15 protofilaments in the transcellular bundles of late pupal Drosophila wing epidermal cells. The microtubules make end-on contact with cell surfaces. Most microtubules in each bundle exhibited a uniform polarity. They were oriented with their minus ends associated with their hemidesmosomal anchorage points at the apical cuticle-secreting surfaces of the cells. Plus ends were directed towards, and were sometimes connected to, basal attachment desmosomes at the opposite ends of the cells.The orientation of microtubules at cell apices, with minus ends directed towards the cell surface, is opposite to the polarity anticipated for microtubules which have elongated centrifugally from centrosomes. It is consistent, however, with evidence that microtubule assembly is nucleated by plasma membrane-associated sites at the apical surfaces of the cells (Mogensen, M. M., and J. B. Tucker. 1987. J. Cell Sci. 88:95-107) after these cells have lost their centriole-containing, centrosomal, microtubule-organizing centers

This report provides evidence for two functionally and spatially distinct centrosomal domains in certain mouse cochlear epithelial cells. The vast majority of microtubules elongate from sites associated with the apical cell surface in these cells rather than from pericentriolar material surrounding the immediate environs of their apically situated centrioles. The distribution of g-tubulin and pericentrin at cell apices has been examined while microtubule nucleation is progressing because these centrosomal proteins are believed to be essential for microtubule nucleation.Antibodies to both proteins bind to pericentriolar regions but no binding has been detected at the apical cell surface-associated sites where the ends of thousands of recently nucleated microtubules are concentrated. Sparse transient microtubule populations can be detected between pericentriolar regions and surface sites while microtubule assembly advances. A procedure apparently operates in which the pericentriolar region functions as a microtubule-nucleating domain and the cell surface-associated sites operate as docking domains which capture the minus ends of microtubules that migrate to them shortly after nucleation.Docking domains may include some components of the pericentriolar material that have been relocated at the cell apex. A docking element hypothesis for centrosomal control of minus end positioning and dynamics in animal cells generally is proposed.This investigation has also shown that the concentration of g-tubulin and pericentrin around centrioles differs spatially and quantitatively in ways that are characteristic for the four cell types studied. Some of these characteristics can be

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Taxanes remain one of the most effective medical treatments for breast cancer. Clinical trials have coupled taxanes with immune checkpoint inhibitors in patients with triple-negative breast cancer (TNBC) with promising results. However, the mechanism linking taxanes to immune activation is unclear. To determine if paclitaxel could elicit an antitumoral immune response, we sampled tumor tissues from patients with TNBC receiving weekly paclitaxel (80 mg/m2) and found increased stromal tumor-infiltrating lymphocytes and micronucleation over baseline in three of six samples. At clinically relevant concentrations, paclitaxel can induce chromosome missegregation on multipolar spindles during mitosis. Consequently, post-mitotic cells are multinucleated and contain micronuclei, which often activate cyclic GMP-AMP synthase (cGAS) and may induce a type I IFN response reliant on the stimulator of IFN genes (STING) pathway. Other microtubule-targeting agents, eribulin and vinorelbine, recapitulate this cGAS/STING response and increased the expression of immune checkpoint molecule, PD-L1, in TNBC cell lines. To test the possibility that microtubule-targeting agents sensitize tumors that express cGAS to immune checkpoint inhibitors, we identified 10 patients with TNBC treated with PD-L1 or PD-1, seven of whom also received microtubule-targeting agents. Elevated baseline cGAS expression significantly correlated with treatment response in patients receiving microtubule-targeting agents in combination with immune checkpoint inhibitors. Our study identifies a mechanism by which microtubule-targeting agents can potentiate an immune response in TNBC. Further, baseline cGAS expression may predict patient treatment response to therapies combining microtubule-targeting agents and immune checkpoint inhibitors.

In this paper we describe a new type of non-centrosomal microtubule-organising centre (MTOC), which is induced by cold treatment of certain cultured Drosophila cells and allows rapid reassembly of microtubule (MT) arrays. Prolonged cooling of two types of cultured Drosophila cells, muscle cells in primary culture and a wing imaginal disc cell line Cl.8+ results in disassembly of MT arrays and induces the formation of clusters of short MTs that have not been described before. Upon rewarming, the clusters are lost and the MT array is re-established within 1 h. In Cl.8+ cells, gamma-tubulin-containing centrosomes are detected, both in cell extensions and in the expected juxtanuclear position, and gamma-tubulin co-localises with the cold-induced MT clusters. The MT plus-end-binding protein, Drosophila EB1, decorates growing tips of MTs extending from clusters. We conclude that the cold-induced MT clusters represent acentrosomal MTOCs, allowing rapid reassembly of MT arrays following exposure to cold.

The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25–30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.