Hec1 Phosphorylation Research Articles

Attenuated Chromosome Oscillation as a Cause of Chromosomal Instability in Cancer Cells.

Simple SummaryChromosomal instability (CIN), a condition in which chromosome missegregation occurs at high rates, is widely seen in cancer cells. Causes of CIN in cancer cells are not fully understood. A recent report suggests that chromosome oscillation, an iterative chromosome motion typically seen in metaphase around the spindle equator, is attenuated in cancer cells, and is associated with CIN. Chromosome oscillation promotes the correction of erroneous kinetochore-microtubule attachments through phosphorylation of Hec1, a kinetochore protein that binds to microtubules, by Aurora A kinase residing on the spindle. In this review, we focused on this unappreciated link between chromosome oscillation and CIN.Chromosomal instability (CIN) is commonly seen in cancer cells, and related to tumor progression and poor prognosis. Among the causes of CIN, insufficient correction of erroneous kinetochore (KT)-microtubule (MT) attachments plays pivotal roles in various situations. In this review, we focused on the previously unappreciated role of chromosome oscillation in the correction of erroneous KT-MT attachments, and its relevance to the etiology of CIN. First, we provided an overview of the error correction mechanisms for KT-MT attachments, especially the role of Aurora kinases in error correction by phosphorylating Hec1, which connects MT to KT. Next, we explained chromosome oscillation and its underlying mechanisms. Then we introduced how chromosome oscillation is involved in the error correction of KT-MT attachments, based on recent findings. Chromosome oscillation has been shown to promote Hec1 phosphorylation by Aurora A which localizes to the spindle. Finally, we discussed the link between attenuated chromosome oscillation and CIN in cancer cells. This link underscores the role of chromosome dynamics in mitotic fidelity, and the mutual relationship between defective chromosome dynamics and CIN in cancer cells that can be a target for cancer therapy.

Chromosome oscillation promotes Aurora A-dependent Hec1 phosphorylation and mitotic fidelity.

Most cancer cells show chromosomal instability, a condition where chromosome missegregation occurs frequently. We found that chromosome oscillation, an iterative chromosome motion during metaphase, is attenuated in cancer cell lines. We also found that metaphase phosphorylation of Hec1 at serine 55, which is mainly dependent on Aurora A on the spindle, is reduced in cancer cell lines. The Aurora A-dependent Hec1-S55 phosphorylation level was regulated by the chromosome oscillation amplitude and vice versa: Hec1-S55 and -S69 phosphorylation by Aurora A is required for efficient chromosome oscillation. Furthermore, enhancement of chromosome oscillation reduced the number of erroneous kinetochore-microtubule attachments and chromosome missegregation, whereas inhibition of Aurora A during metaphase increased such errors. We propose that Aurora A-mediated metaphase Hec1-S55 phosphorylation through chromosome oscillation, together with Hec1-S69 phosphorylation, ensures mitotic fidelity by eliminating erroneous kinetochore-microtubule attachments. Attenuated chromosome oscillation and the resulting reduced Hec1-S55 phosphorylation may be a cause of CIN in cancer cell lines.

Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy

Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is correlated with cancer formation, progression, and survival. Phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function, thus any disruption of Hec1/Nek2 protein–protein interaction has potential for cancer therapy. We have developed T-1101 tosylate (9j tosylate, 9j formerly known as TAI-95), optimized from 4-aryl-N-pyridinylcarbonyl-2-aminothiazole of scaffold 9 by introducing various C-4’ substituents to enhance potency and water solubility, as a first-in-class oral clinical candidate for Hec1 inhibition with potential for cancer therapy. T-1101 has good oral absorption, along with potent in vitro antiproliferative activity (IC50: 14.8–21.5 nM). It can achieve high concentrations in Huh-7 and MDA-MB-231 tumor tissues, and showed promise in antitumor activity in mice bearing human tumor xenografts of liver cancer (Huh-7), as well as of breast cancer (BT474, MDA-MB-231, and MCF7) with oral administration. Oral co-administration of T-1101 halved the dose of sorafenib (25 mg/kg to 12.5 mg/kg) required to exhibit comparable in vivo activity towards Huh-7 xenografts. Cellular events resulting from Hec1/Nek2 inhibition with T-1101 treatment include Nek2 degradation, chromosomal misalignment, and apoptotic cell death. A combination of T-1101 with either of doxorubicin, paclitaxel, and topotecan in select cancer cells also resulted in synergistic effects. Inactivity of T-1101 on non-cancerous cells, a panel of kinases, and hERG demonstrates cancer specificity, target specificity, and cardiac safety, respectively. Subsequent salt screening showed that T-1101 tosylate has good oral AUC (62.5 μM·h), bioavailability (F = 77.4%), and thermal stability. T-1101 tosylate is currently in phase I clinical trials as an orally administered drug for cancer therapy.

Assessing the Role of Ndc80 Kinetochore Complex in Accurate Chromosome Segregation

Mitosis is required to grow and develop from a single cell to a multicellular organism. However, this process can often lead to cancer when its regulation is lost. The Highly Express in Cancer 1 (Hec1) protein is one of the four subunits of the Nuclear Division Cycle (Ndc80) complex present in mitosis. The function of this complex is required for robust kinetochore‐microtubule (kMT) attachment and is regulated by Aurora B Kinase phosphorylation of Hec1. Given that Hec1 protein binds directly to spindle microtubules, it certainly plays a critical role in chromosome alignment and segregation. To assess how Hec1 leads to defects in cell division cycle in human cells and induce cancer, we expressed a non‐phosphorylatable version of Hec1 (Hec1‐9A) in MDA‐MB‐436 (breast cancer) cells. We observed how mitosis was modified by the Hec1‐9A mutant by employing immunofluorescence microscopy to mark the chromosomes and the mitotic spindle in our Hec1‐9A positive and control cells. The Hec1‐9A satisfied the genome surveillance mechanism, thus, allowing cells that presented erroneous chromosome alignment and segregation to exit mitosis. Further studies will be aimed to investigate whether spindle checkpoint is defective at erroneous kMT attachments and whether Hec1 plays a role in other steps of mitosis.Support or Funding InformationFunded by NIH‐NIGMS #2R25GM096955Funded by NIH #R00CA178188This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic acetylation of the kinetochore-associated protein HEC1 ensures accurate microtubule–kinetochore attachment

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.

Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore-microtubule dynamics.

Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.

Abstract A38: In vitro and in vivo characterization of a novel NEK2 inhibitor MBM5 for the treatment of gastrointestinal cancer

Abstract NEK2 is a conserved mitotic regulator critical for centrosome splitting and correct chromosome segregation. Aberrant expression of NEK2 has been found in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. Here we report the discovery of a novel small molecule NEK2 inhibitor, MBM5, for the treatment of gastrointestinal cancer cells. A series of compounds have been synthesized to determine their potency against NEK2. Through in vitro kinase assay, we found that compound MBM5 exhibited a significant inhibitory effect on the kinase activity of NEK2. MBM5 also demonstrated strong inhibition on proliferation of a broad spectrum of tumor cells, especially gastrointestinal cancer cells (IC50<1 μM). Treating cells with MBM5 triggered a significant accumulation of cells at G2/M phase and a modest accumulation of cells with >4N DNA content. By releasing cells from the G1/S boundary, we found that the progression from G2/M to G1 phases were completely abrogated in the presence of MBM5. In consistence, we further observed that MBM5 induced chromosomal misalignment in metaphase and slightly increased the level of multinucleated cells, confirming the perturbed mitotic progression. And these was correlated with MBM5-inhibited phosphorylation of Hec1 and Histone 3. Over time, the accumulation of chromosomal abnormalities led to apoptosis, as evidenced by the increased population of Annexin-V/PI positive cells, the up-regulated expression of cleaved-caspase 3 and the cleavage of PARP. In vivo daily treatment with MBM5 (ip, 20 mg/kg, 3 weeks) significantly inhibited the growth of MGC-803 gastric tumor and HCT-116 colonal tumor xenografts. Altogether, this study demonstrates that MBM5 interfered with NEK2 functions both in cells and tumor xenografts, thus showing its potential in anti-cancer treatment regimens. Citation Format: Yanfen Fang, Xiongwen Zhang, Wenhao Hu, Yannan Kong, Hong Wang, Mengli Zhu. In vitro and in vivo characterization of a novel NEK2 inhibitor MBM5 for the treatment of gastrointestinal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A38.

Accurate phosphoregulation of kinetochore-microtubule affinity requires unconstrained molecular interactions.

Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore-MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore-MT affinity. In contrast, modeling the kinetochore interface with repetitive MT binding sites predicts a switchlike response. To reconcile these findings, we hypothesize that interactions between NDC80 complexes and MTs are not constrained, i.e., the NDC80 complexes can alternate their binding between adjacent kinetochore MTs. Experiments using cells with phosphomimetic Hec1 mutants corroborate predictions of such a model but not of the repetitive sites model. We propose that accurate regulation of kinetochore-MT affinity is driven by incremental phosphorylation of an NDC80 molecular "lawn," in which the NDC80-MT bonds reorganize dynamically in response to the number and stability of MT attachments.

Novel small molecules disrupting Hec1/Nek2 interaction ablate tumor progression by triggering Nek2 degradation through a death-trap mechanism

Hec1 (highly expressed in cancer 1) or Nek2 (NIMA-related kinase 2) is often overexpressed in cancers with poor prognosis. Both are critical mitotic regulators, and phosphorylation of Hec1 S165 by Nek2 is required for proper chromosome segregation. Therefore, inactivation of Hec1 and Nek2 by targeting their interaction with small molecules represents an ideal strategy for tackling these types of cancers. Herewe showed that new derivatives of INH (inhibitor for Nek2 and Hec1 binding) bind to Hec1 at amino acids 394-408 on W395, L399 and K400 residues, effectively blocking Hec1 phosphorylation on S165 by Nek2, and killing cancer cells at the nanomolar range. Mechanistically, the D-box (destruction-box) region of Nek2 specifically binds to Hec1 at amino acids 408-422, immediately adjacent to the INH binding motif. Subsequent binding of Nek2 to INH-bound Hec1 triggered proteasome-mediated Nek2 degradation, whereas the Hec1 binding defective Nek2 mutant, Nek2 R361L, resisted INH-induced Nek2 degradation. This finding unveils a novel drug-action mechanism where the binding of INHs to Hec1 forms a virtual death-trap to trigger Nek2 degradation and eventually cell death. Furthermore, analysis of the gene expression profiles of breast cancer patient samples revealed that co-elevated expressions of Hec1 and Nek2 correlated with the shortest survival. Treatment of mice with this kind of tumor with INHs significantly suppressed tumor growth without obvious toxicity. Taken together, the new INH derivatives are suitable for translation into clinical application.

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.

Chk1 and Mps1 jointly regulate correction of merotelic kinetochore attachments.

If uncorrected, merotelic kinetochore attachments can induce mis-segregated chromosomes in anaphase. We show that checkpoint kinase 1 (Chk1) protects vertebrate cells against merotelic attachments and lagging chromosomes and is required for correction of merotelic attachments during a prolonged metaphase. Decreased Chk1 activity leads to hyper-stable kinetochore microtubules, unstable binding of MCAK, Kif2b and Mps1 to centromeres or kinetochores and reduced phosphorylation of Hec1 by Aurora-B. Phosphorylation of Aurora-B at serine 331 (Ser331) by Chk1 is high in prometaphase and decreases significantly in metaphase cells. We propose that Ser331 phosphorylation is required for optimal localization of MCAK, Kif2b and Mps1 to centromeres or kinetochores and for Hec1 phosphorylation. Furthermore, inhibition of Mps1 activity diminishes initial recruitment of MCAK and Kif2b to centromeres or kinetochores, impairs Hec1 phosphorylation and exacerbates merotelic attachments in Chk1-deficient cells. We propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b and Hec1 to correct merotelic attachments. These results suggest a role for Chk1 and Mps1 in error correction.

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

Precise control of the attachment strength between kinetochores and spindle microtubules is essential to preserve genomic stability. Aurora B kinase has been implicated in regulating the stability of kinetochore-microtubule attachments but its relevant kinetochore targets in cells remain unclear. Here, we identify multiple serine residues within the N-terminus of the kinetochore protein Hec1 that are phosphorylated in an Aurora-B-kinase-dependent manner during mitosis. On all identified target sites, Hec1 phosphorylation at kinetochores is high in early mitosis and decreases significantly as chromosomes bi-orient. Furthermore, once dephosphorylated, Hec1 is not highly rephosphorylated in response to loss of kinetochore-microtubule attachment or tension. We find that a subpopulation of Aurora B kinase remains localized at the outer kinetochore even upon Hec1 dephosphorylation, suggesting that Hec1 phosphorylation by Aurora B might not be regulated wholly by spatial positioning of the kinase. Our results define a role for Hec1 phosphorylation in kinetochore-microtubule destabilization and error correction in early mitosis and for Hec1 dephosphorylation in maintaining stable attachments in late mitosis.

Phosphorylation of the Mitotic Regulator Protein Hec1 by Nek2 Kinase Is Essential for Faithful Chromosome Segregation

Hec1 (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 mutant changing Ser(201) to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.