Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

<p>Abstract</p> <p>Background</p> <p>The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown.</p> <p>Results</p> <p>We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.</p> <p>Conclusion</p> <p>Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.</p

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

Molecular mechanisms of cellular stress responses in cancer and their therapeutic implications

In response to stress, cells can activate a myriad of signalling pathways to bring about a specific cellular outcome, including cell cycle arrest, DNA repair, senescence and apoptosis. This response is pivotal for tumour suppression as all of these outcomes result in restriction of the growth and/or elimination of damaged and pre-malignant cells. Thus, a large number of anti-cancer agents target specific components of stress response signalling pathways with the aim of causing tumour regression by stimulating cell death. However, the efficacy of these agents is often impaired due to mutations in genes that are involved in these stress-responsive signalling pathways and instead the oncogenic potential of a cell is increased leading to the initiation and/or progression of tumourigenesis. Moreover, these genetic defects can increase or contribute to resistance to chemotherapeutic agents and/or radiotherapy. Modulating the outcome of cellular stress responses towards cell death in tumour cells without affecting surrounding normal cells is thus one of the ultimate aims in the development of new cancer therapeutics. To achieve this aim, a detailed understanding of cellular stress response pathways and their aberrations in cancer is required.This Research topic aims to reflect the broadness and complexity of this important area of cancer research

Sorting Nexin 9 Recruits Clathrin Heavy Chain to the Mitotic Spindle for Chromosome Alignment and Segregation

<div><p>Sorting nexin 9 (SNX9) and clathrin heavy chain (CHC) each have roles in mitosis during metaphase. Since the two proteins directly interact for their other cellular function in endocytosis we investigated whether they also interact for metaphase and operate on the same pathway. We report that SNX9 and CHC functionally interact during metaphase in a specific molecular pathway that contributes to stabilization of mitotic spindle kinetochore (K)-fibres for chromosome alignment and segregation. This function is independent of their endocytic role. SNX9 residues in the clathrin-binding low complexity domain are required for CHC association and for targeting both CHC and transforming acidic coiled-coil protein 3 (TACC3) to the mitotic spindle. Mutation of these sites to serine increases the metaphase plate width, indicating inefficient chromosome congression. Therefore SNX9 and CHC function in the same molecular pathway for chromosome alignment and segregation, which is dependent on their direct association.</p> </div

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

<p>Abstract</p> <p>Background</p> <p>During metaphase clathrin stabilises the mitotic spindle kinetochore(K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis.</p> <p>Results</p> <p>Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition.</p> <p>Conclusions</p> <p>Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.</p

SNX9 is required for efficient recruitment of CHC and TACC3 to the mitotic spindle.

<p>(A) Representative microscopy images illustrating the localization of CHC during interphase (Int) and the indicated mitotic stages in untreated cells and cells depleted of SNX9, SNX18 or SNX33 by siRNA. Met, metaphase. Ana, anaphase. Cyto, cytokinesis. Scale bars represent 10 µm. (B–C) The graphs represents the fluorescence intensity ratio of CHC at the mitotic spindle over the whole cell (B) and the overall fluorescence intensity of CHC within the whole cell (C) during metaphase (mean ± S.E.M., n > 16 per sample). ns, not significant; ***, <i>p</i> < 0.001 (One-way ANOVA). (D) Representative microscopy images demonstrating the localization of SNX9 (upper panels) in untreated and CHC-depleted HeLa cells during interphase, and metaphase. DNA shown in lower panels. Scale bars represent 10 µm. (E–F) Untreated, SNX9 and CHC-depleted metaphase HeLa cells were stained for TACC3. Representative microscopy images of TACC3 localization (upper panels) in these cells are shown in D. DNA shown in lower panels. Graphs represents the amount of TACC3 in each cell, expressed as (F) ratio of fluorescence intensity at the mitotic spindle compared to the whole cell and (G) the overall fluorescence intensity of TACC3 within the whole cell (mean ± S.E.M., n = > 6).</p

CHC interaction with the LC domain of SNX9 is required for efficient recruitment of mitotic spindle components.

<p>(A) A schematic diagram illustrating the domain structure of SNX9 and the amino acids in part of the low complexity sequence (LC domain). SNX9 contains an Src-homology 3 (SH3) domain at the N terminus followed by a low complexity (LC) domain, a phox-homology (PX) domain that is flanked by Yoke (Y) domains. A C-terminal Bin/Amphiphysin/Rvs (BAR) domain is located at the C-termini. Single and double mutations in LC1 (LC1 and LC1W1) and LC2 (LC2 and LC2W1) regions are shown whereby the tryptophan (W) residues (underlined) were mutated to serine (S). (B) GST alone, full length wild-type GST-SNX9 (WT) and GST-SNX9 harbouring LC1, LC1W1, LC2 and LC2W1 mutants coupled to glutathione-Sepharose were incubated with lysates from asynchronously growing HeLa cells and immunoblotted for CHC, dynII and dynII^S764. Lower panel shows amount of GST fusion protein in each sample (10%) as determined by Coomassie Blue staining. Lysate (2.5%) was also immunoblotted for the above mentioned proteins to reveal input. (C) The amount of CHC bound to the GST-SNX9 proteins indicated in B were quantified by densitometry analyses of immunoblots. Graph illustrates the relative amount of CHC bound to mutant GST-SNX9 (mean ± S.E.M. from 3–4 independent experiments) compared to wild-type GST-SNX9. (D–G) Metaphase-synchronised HeLa cells expressing GFP alone or GFP-SNX9-WT, LC1 or LC2 mutants were stained for CHC and TACC3. Representative microscopy images of the localization of CHC (D) and TACC3 (F) in these cells are shown. Scale bars represent 10 µm. The graphs represent the fluorescence intensity ratio of CHC (E) and TACC3 (G) at the mitotic spindle over the whole cell. (H) The graph illustrates the width of the metaphase plate of those cells analysed in E and G. Data shown in graphs (E, G and H) represent mean ± S.E.M. from at least two independent experiments. n = 15-30 cells per sample in each experiment. ns, not significant; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 (One-way ANOVA).</p

SNX9 and CHC are required for MT nucleation and K-fibre organization at the mitotic spindle.

<p>(A–B) Untreated HeLa cells and HeLa cells transfected with SNX9 or CHC siRNA, were subjected to a MT nucleation assay as described in methods section. Representative microscopy images illustrating MT regrowth from the spindle poles after the time period (A). Graphs show the length of the longest MT (mean ± S.E.M.) at each time point (n=11-19 cells per sample from two independent experiments; B). (C) Representative microscopy images of a HURP stained untreated, SNX9-depleted and CHC-depleted HeLa cell in metaphase, to highlight K-fibres (upper panels). DNA shown in lower panels. (D) The graph represents the fluorescence intensity ratio of HURP at the mitotic spindle over the whole cell. Scale bars represent 10 µm. ns, not significant; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 (One-way ANOVA). (E) The graph (mean ± S.E.M. from three independent experiments) shows the width of the centre of the metaphase plate of SNX9 and CHC-depleted HeLa cells compared to that in untreated HeLa cells in metaphase.</p