Mitotic Centromere-associated Kinesin Research Articles

Abstract 6598: Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors

Abstract The activation of STING (stimulator of interferon genes) has been widely shown to induce signaling pathways, triggering type I interferon and ultimately generating anti-tumor immunity. Whereas initial preclinical studies on STING agonists exhibited boost in anti-tumor response, clinical trials combining STING agonist and immune checkpoint blockade agent PD-1 reported that only small subset of patients benefited from the treatment. This study highlights a potential key mechanism underlying the lack of benefit from STING agonist observed in most clinical trial patients. We reveal a phenomenon known as chromosomal instability (CIN), characterized by persistent chromosome segregation errors over multiple rounds of mitosis, drives chronic activation of cGAS (cyclic GMP‒AMP synthase) through continuously supplying its substrate in the form of micronuclei. Chronic activation of cGAS stimulates degradation of its downstream target STING in a feedback-loop manner. This consequently attenuates type I interferon signaling upon STING activation, blunting its anti-tumor response. Remarkably, we show suppression of CIN, achieved through introduction of mitotic centromere-associated kinesin (MCAK), or inhibition of cGAS activity, either by pharmacological agent or total genetic ablation of cGAS using CRISPR-Cas9, restores type I interferon production and anti-tumor immunity upon the administration of STING agonist, MSA-2 in four of the five advanced tumor models. Furthermore, we show this restoration of type I interferon signaling and anti-tumor immunity is cancer cell STING-dependent as genetic deletion of STING in the tumor cells nullifies this effect. In summary, we provide a mechanistic insight underlying the limited anti-tumor response observed in STING agonist trials and propose potential strategies, through finetuning of CIN level or inhibition of cGAS activity, to illicit stronger anti-tumor immunity in combination with STING agonist treatment. Citation Format: Christy Hong, Lindsay Caprio, Mercedes Duran, Meri Rogava, Hsiang-His Ling, Sreeharsha Gurrapu, Leon Ebel, Melody Di Bona, Benjamin Izar, Samuel F. Bakhoum. Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6598.

Abstract 5360: Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade

Abstract Non-small cell lung cancers (NSCLCs) harboring deletions or inactivating mutations in STK11 (encoding LKB1) are associated with poor prognosis and immune evasion, but the underlying mechanisms are poorly understood. Through integrative analyses of >10,000 molecular profiles from public data sources and an additional ~15,000 new patient data profiles, we find identify STK11-deficient NSCLC as an archetypical chromosomally unstable tumor. Using high-content imaging, we show that both human and murine STK11-mutant models have a higher rate of micronuclei and chromosome mis-segregation events, confirming their high degree of chromosomal instability (CIN). We show that CIN-induced tonic activation of the cGAS-STING pathway results in impaired type I interferon expression, thus, turning the physiological function of this pathway on its head. We show that temporary relief of tonic CIN-induced signaling reprogrammed cancer cells towards production of type I interferons. We achieved this through orthogonal routes, including genetic suppression of CIN by overexpression of mitotic centromere-associated kinesin (MCAK), rescue of wild-type STK11 (but not its kinase-dead) allele, genetic deletion or pharmacological inhibition of cGAS. Relief through any of these approaches re-sensitized cells to acute STING agonism with its endogenous ligand (cGAMP) or pharmacological stimulation. Using otherwise isogenic, syngeneic KRAS-mutant Stk11/Lkb1-/- (KL) and KRAS-mutant p53 mutant (KP) murine models, we show that cGAS KO sensitized otherwise highly resistant KL models to immune checkpoint blockade (ICB), while an increase in CIN (through overexpression of dominant-negative MCAK) in KP conferred resistance in this otherwise ICB-sensitive model. In summary, we define STK11-mutant as archetypical chromosomally unstable form of NSCLC, provide mechanistic basis that correlates with poor clinical outcomes, and demonstrate how relief of tonic CIN-induced changes may be therapeutically leveraged. Citation Format: Lindsay A. Caprio, Christy Hong, Amit D. Amin, Somnath Tagore, Mercedes Duran, Meri Rogava, Luke Cai, Leon Ebel, Johannes Melms, Hanina Hibshoosh, Samuel Bakhoum, Benjamin Izar. Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5360.

EGFR-mediated hyperacetylation of tubulin induced docetaxel resistance by downregulation of HDAC6 and upregulation of MCAK and PLK1 in prostate cancer cells.

Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for castration-resistant prostate cancer (PCa). However, clinical treatment with docetaxel often encounters a number of undesirable effects, including drug resistance. Tubulin isoforms have been previously examined for their resistance to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, a series of docetaxel-resistant PC/DX cell sublines were established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Western blotting results showed significantly higher expression of acetyl-tubulin, α-tubulin, β-tubulin, γ-tubulin, and βIII-tubulin in PC/DX25 than in parental PC3 cells. PC/DX25 with greater resistance to docetaxel had higher levels of acetyl-tubulin and mitotic centromere-associated kinesin (MCAK) than PC3 cells. This study found that docetaxel induced the expression of acetyl-tubulin and MCAK in PC3 cells at a dose- and time-dependent manner. Both mRNA and protein levels of histone deacetylase 6 (HDAC6) were significantly decreased in PC/DX25 compared with PC3 cells. PC3 increased the resistance to docetaxel by HDAC6 knockdown and Tubastatin A (HDAC6 inhibitor). Conversely, PC/DX25 reversed the sensitivity to docetaxel by MCAK knockdown. Notably, flow cytometry analysis revealed that MCAK knockdown induced significantly sub G1 fraction in PC/DX cells. Overexpression of polo-like kinase-1 increased the cell survival rate and resistance to docetaxel in PC3 cells. Moreover, epidermal growth factor receptor (EGFR) activation induced the upregulation of acetyl-tubulin in docetaxel-resistant PCa cells. These findings demonstrated that the EGFR-mediated upregulated expression of acetyl-tubulin played an important role in docetaxel-resistant PCa.

Ablation of KIF2C in Purkinje cells impairs metabotropic glutamate receptor trafficking and motor coordination in male mice.

Kinesin family member 2C (KIF2C)/mitotic centromere-associated kinesin (MCAK), is thought to be oncogenic as it is involved in tumour progression and metastasis. Moreover, it also plays a part in neurodegenerative conditions like Alzheimer's disease and psychiatric disorders such as suicidal schizophrenia. Our previous study conducted on mice demonstrated that KIF2C is widely distributed in various regions of the brain, and is localized in synaptic spines. Additionally, it regulates microtubule dynamic properties through its own microtubule depolymerization activity, thereby affecting AMPA receptor transport and cognitive behaviour in mice. In this study, we show that KIF2C regulates the transport of mGlu1 receptors in Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells results in abnormal gait, reduced balance ability and motor incoordination in male mice. These data suggest that KIF2C is essential for maintaining normal transport and synaptic function of mGlu1 and motor coordination in mice. KEY POINTS: KIF2C is localized in synaptic spines of hippocampus neurons, and regulates excitatory transmission, synaptic plasticity and cognitive behaviour. KIF2C is extensively expressed in the cerebellum, and we investigated its functions in development and synaptic transmission of cerebellar Purkinje cells. KIF2C deficiency in Purkinje cells alters the expression of metabotropic glutamate receptor 1 (mGlu1) and the AMPA receptor GluA2 subunit at Purkinje cell synapses, and changes excitatory synaptic transmission, but not inhibitory transmission. KIF2C regulates the transport of mGlu1 receptors in Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells affects motor coordination, but not social behaviour in male mice.

Abstract 5944: Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade

Abstract Non-small cell lung cancers (NSCLCs) harboring deletions or inactivating mutations in STK11 (encoding LKB1) are associated with treatment-resistance, including to immune checkpoint blockade, and poor survival, yet the underlying mechanisms are poorly understood. Here, we combined multi-modal single-cell transcriptomics, whole-genome sequencing (WGS), and analysis of public data bases totaling >10,000 NSCLC whole-exome sequencing (WES) and when available RNA (RNA-seq) profiles of AACR GENIE, MSK IMPACT, TCGA and CPTAC, high-content imaging, and functional assays, to determine genomic and mechanistic features of STK11-mutant NSCLC. Across human WES/RNA-seq data, we find that STK11-mutant NSCLC have a significantly higher fraction of genome altered (FGA) and score strongly for the mRNA-based CIN70 signature compared to other NSCLC genotypes, overall indicating that these tumors have a higher degree of chromosomal instability (CIN). Using high-content imaging, we show that both human and murine STK11-mutant models have a higher rate of micronuclei and chromosome-mis-segregation events, confirming their CINhigh status in dynamic assays. Next, we show that tonic CIN-induced activation of cGAS-STING signaling, whose activation is typically thought of inducing type I interferon expression, results in strong suppression of anti-tumor immune signaling. Remarkably, genetic or pharmacologic suppression of cGAS, and thus depletion of the STING ligand cGAMP, results in reprogramming and re-sensitization of STK11-mutant models to cGAS-STING mediated type I interferon responses. Furthermore, we show that CIN promotes excessive production cGAMP in STK11-mutant models. We find upregulation of two ectonucleotidases (ENPP1 and CD73/NT5E) that hydrolyze exported cGAMP to adenosine, which may further contribute to the highly dysfunctional tumor-microenvironment observed in STK11-mutant tumors. Lastly, suppression of CIN through overexpression of mitotic-centromere associated kinesin (MCAK) in murine STK11-mutant models results in decreased tumor growth and sensitization to anti-PD1 therapy. In summary, we define STK11-mutant as archetypical chromosomally form of NSCLC, provide mechanistic basis that corelates with poor clinical outcomes, and demonstrate how relief of tonic CIN-induced changes may be therapeutically leveraged. Citation Format: Lindsay A. Caprio, Christy Hong, Amit Dipak Amin, Somnath Tagore, Johannes Melms, Luke Cai, Yiping Wang, Patricia Ho, Michael Mu, Hanina Hibshoosh, Brian Henick, Kwok K. Wong, Samuel F. Bakhoum, Benjamin Izar. Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5944.

Septins guide noncentrosomal microtubules to promote focal adhesion disassembly in migrating cells.

Endothelial cell migration is critical for vascular angiogenesis and is compromised to facilitate tumor metastasis. The migratory process requires the coordinated assembly and disassembly of focal adhesions (FA), actin, and microtubules (MT). MT dynamics at FAs deliver vesicular cargoes and enhance actomyosin contractility to promote FA turnover and facilitate cell advance. Noncentrosomal (NC) MTs regulate FA dynamics and are sufficient to drive cell polarity, but how NC MTs target FAs to control FA turnover is not understood. Here, we show that Rac1 induces the assembly of FA-proximal septin filaments that promote NC MT growth into FAs and inhibit mitotic centromere-associated kinesin (MCAK)-associated MT disassembly, thereby maintaining intact MT plus ends proximal to FAs. Septin-associated MT rescue is coupled with accumulation of Aurora-A kinase and cytoplasmic linker-associated protein (CLASP) localization to the MT between septin and FAs. In this way, NC MTs are strategically positioned to undergo MCAK- and CLASP-regulated bouts of assembly and disassembly into FAs, thereby regulating FA turnover and cell migration.

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover.

Simple SummaryThe microtubule (MT) cytoskeleton is a crucial factor for organized cell motility and migration of cancer as well as benign cells. Mitotic centromere-associated kinesin (MCAK/KIF2C) is a member of the kinesin-13 family, which is important for the regulation of MT dynamics. Its overexpression has been reported to be related to increased metastasis in various tumor entities. Our study further elucidate how MCAK’s is able to modulate cell migration and invasion. Interfering with the precise regulated expression of MCAK led to impaired FA protein composition and altered their phosphorylation status, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. MCAK regulates these processes by affecting the actin-MT cytoskeleton dynamics, providing molecular mechanisms by which a deregulation of MCAK could promote tumor metastasis.The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

KIF2C promotes the proliferation of hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality and morbidity rates. In recent years, HCC targeted therapy has gained increasing attention. Due to the heterogeneity and high metastasis of HCC, more effective therapeutic targets are needed. Kinesin family member 2C (KIF2C), also known as mitotic centromere-associated kinesin, is a microtubule-based motor protein which is involved in a variety of important cellular processes, such as mitosis. The effects of KIF2C on cancer progression and development have been widely studied; however, its potential effects on HCC remains unclear. In the present study, high expression of KIF2C in human HCC tissues was demonstrated using The Cancer Genome Atlas database and immunohistochemistry assays. KIF2C expression was associated with HCC prognosis, including overall survival and disease-free survival. KIF2C expression was also associated with clinical pathological characteristics including the number of tumor nodes (P=0.015) and tumor size (P=0.009). KIF2C knockdown inhibited the proliferation of HCC cells in vitro, confirmed by MTT and colony formation assays, and suppressed tumor growth in mice which was confirmed by a xenograft mouse model. Together, the results suggested that KIF2C may serve as a promising therapeutic target for the treatment of HCC.

Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration.

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.

CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer.

BackgroundOverexpression of Aurora‐A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora‐A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients. Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.MethodsHere, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237. Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora‐B and mitotic centromere‐associated kinesin (MCAK).ResultsAmong the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237. Mechanistic studies showed that co‐treatment with Aurora‐A and Haspin inhibitors abolished the recruitment of Aurora‐B and mitotic centromere‐associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore‐microtubule (KT‐MT) attachment failure, and severe mitotic catastrophe. We further showed that the combination of MLN8237 and the Haspin inhibitor CHR‐6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.ConclusionsThese findings establish Haspin as a synthetic lethal target and demonstrate CHR‐6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.

Phosphatidylinositol-5-phosphate 4-kinase gamma accumulates at the spindle pole and prevents microtubule depolymerization

BackgroundA previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles.ResultsPIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite.ConclusionsOur current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

Human Mitotic Centromere-Associated Kinesin Is Targeted by MicroRNA 485-5p/181c and Prognosticates Poor Survivability of Breast Cancer.

Purpose This study aims to evaluate the prognostic value of human Mitotic Centromere-Associated Kinesin (MCAK), a microtubule-dependent molecular motor, in breast cancers. The posttranscriptional regulation of MCAK by microRNAs will also be explored. Methods The large-scale gene expression datasets of breast cancer (total n=4,677) were obtained from GEO, NKI, and TCGA database. Kaplan-Meier and Cox analyses were used for survival analysis. MicroRNAs targeting MCAK were predicted by bioinformatic analysis and validated by a dual-luciferase reporter assay. Results The expression of MCAK was significantly associated with aggressive features of breast cancer, including tumor stage, Elston grade, and molecular subtypes, for global gene expression datasets of breast cancer (p<0.05). Overexpression of MCAK was significantly associated with poor outcome in a dose-dependent manner for either ER-positive or ER-negative breast cancer. Evidence from bioinformatic prediction, coexpression assays, and gene set enrichment analyses suggested that miR-485-5p and miR-181c might target MCAK and suppress its expression. A 3'UTR dual-luciferase target reporter assay demonstrated that miR-485-5p and miR-181c mimics specifically inhibited relative Firefly/Renilla luciferase activity by about 50% in corresponding reporter plasmids. Further survival analysis also revealed that miR-485-5p (HR=0.59, 95% CI 0.37-0.92) and miR-181c (HR=0.54, 95% CI 0.34-0.84) played opposite roles of MCAK (HR=2.80, 95% CI 1.77-4.57) and were significantly associated with better outcome in breast cancers. Conclusion MCAK could serve as a prognostic biomarker for breast cancers. miR-485-5p and miR-181c could specifically target and suppress the MCAK gene expression in breast cancer cells.

β-tubulin carboxy-terminal tails exhibit isotype-specific effects on microtubule dynamics in human gene-edited cells.

Microtubules are highly dynamic structures that play an integral role in fundamental cellular functions. Different α- and β-tubulin isotypes are thought to confer unique dynamic properties to microtubules. The tubulin isotypes have highly conserved structures, differing mainly in their C-terminal tail sequences. However, little is known about the importance of the C-terminal tail in regulating and co-ordinating microtubule dynamics. We developed syngeneic human cell models using gene-editing to precisely modify the β-tubulin C-terminal tail region while preserving the endogenous microtubule network. Fluorescent microscopy of live cells, coupled with advanced image analysis revealed that the β-tubulin C-terminal tails differentially co-ordinate the collective and individual dynamic behaviour of microtubules by affecting microtubule growth rates and explorative microtubule assembly in an isotype-specific manner. Furthermore, βI- and βIII-tubulin C-terminal tails differentially regulate the sensitivity of microtubules to tubulin-binding agents and the microtubule depolymerising protein MCAK. The sequence of the β-tubulin tail encodes regulatory information that instructs and co-ordinates microtubule dynamics, thereby fine-tuning microtubule dynamics to support cellular functions.

Tumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level.

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.

MCAK-mediated regulation of endothelial cell microtubule dynamics is mechanosensitive to myosin-II contractility.

Compliance and dimensionality mechanosensing, the processes by which cells sense the physical attributes of the extracellular matrix (ECM), are known to drive cell branching and shape change largely through a myosin-II-mediated reorganization of the actin and microtubule (MT) cytoskeletons. Subcellular regulation of MT dynamics is spatially controlled through a Rac1-Aurora-A kinase pathway that locally inhibits the MT depolymerizing activity of mitotic centromere-associated kinesin (MCAK), thereby promoting leading-edge MT growth and cell polarization. These results suggest that the regulation of MT growth dynamics is intimately linked to physical engagement of the cell with the ECM. Here, we tested the hypothesis that MCAK contributes to compliance and dimensionality mechanosensing-mediated regulation of MT growth dynamics through a myosin-II-dependent signaling pathway. We cultured endothelial cells (ECs) on collagen-coupled stiff or compliant polyacrylamide ECMs to examine the effects of MCAK expression on MT growth dynamics and EC branching morphology. Our results identify that MCAK promotes fast MT growth speeds in ECs cultured on compliant 2D ECMs but promotes slow MT growth speeds in ECs cultured on compliant 3D ECMs, and these effects are myosin-II dependent. Furthermore, we find that 3D ECM engagement uncouples MCAK-mediated regulation of MT growth persistence from myosin-II-mediated regulation of growth persistence specifically within EC branched protrusions.

Aurora kinase B dependent phosphorylation of 53BP1 is required for resolving merotelic kinetochore-microtubule attachment errors during mitosis.

Defects in resolving kinetochore-microtubule attachment mistakes during mitosis is linked to chromosome instability associated with carcinogenesis as well as resistance to cancer therapy. Here we report for the first time that tumor suppressor p53-binding protein 1 (53BP1) is phosphorylated at serine 1342 (S1342) by Aurora kinase B both in vitro and in human cells, which is required for optimal recruitment of 53BP1 at kinetochores. Furthermore, 53BP1 staining normally localized on the outer kinetochore, extended to the whole kinetochore when it is merotelically-attached, in concert with mitotic centromere-associated kinesin. Kinetochore-binding of pS1342-53BP1 is essential for efficient resolving of merotelic attachment, a spontaneous kinetochore-microtubule connection error that usually causes aneuploidy. Consistently, loss of 53BP1 results in significant increase in lagging chromosome events, micronuclei formation and aneuploidy, due to the unresolved merotely in both cancer and primary cells, which is prevented by ectopic wild type 53BP1 but not by the nonphophorylable S1342A mutant. We thus document a novel DNA damage-independent function of 53BP1 in maintaining faithful chromosome segregation during mitosis.

GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK.

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.

The Ska complex promotes Aurora B activity to ensure chromosome biorientation.

Chromosome biorientation and accurate segregation rely on the plasticity of kinetochore-microtubule (KT-MT) attachments. Aurora B facilitates KT-MT dynamics by phosphorylating kinetochore proteins that are critical for KT-MT interactions. Among the substrates whose microtubule and kinetochore binding is curtailed by Aurora B is the spindle and kinetochore-associated (Ska) complex, a key factor for KT-MT stability. Here, we show that Ska is not only a substrate of Aurora B, but is also required for Aurora B activity. Ska-deficient cells fail to biorient and display chromosome segregation errors underlying suppressed KT-MT turnover. These defects coincide with KNL1-Mis12-Ndc80 network hypophosphorylation, reduced mitotic centromere-associated kinesin localization, and Aurora B T-loop phosphorylation at kinetochores. We further show that Ska requires its microtubule-binding capability to promote Aurora B activity in cells and stimulates Aurora B catalytic activity in vitro. Finally, we show that protein phosphatase 1 counteracts Aurora B activity to enable Ska kinetochore accumulation once biorientation is achieved. We propose that Ska promotes Aurora B activity to limit its own microtubule and kinetochore association and to ensure that KT-MT dynamics and stability fall within an optimal balance for biorientation.

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.