Mitotic Arrest Deficient 1 Research Articles

MAD1 deficiency accelerates hepatocellular proliferation via suppressing TGF-β signaling

Numerous researches have reported on the regulatory network of liver regeneration induced by partial hepatectomy (PH). However, information on key molecules and/or signaling pathways regulating the termination stage of liver regeneration remains limited. In this study, we identify hepatic mitotic arrest deficient 1 (MAD1) as a crucial regulator of transforming growth factor β (TGF-β) in the hepatocyte to repress liver regeneration. MAD1 has a low expression level at the rapid proliferation phase but significantly increases at the termination phase of liver regeneration. We show that MAD1 deficiency accelerates hepatocyte proliferation and enhances mitochondrial biogenesis and respiratory. Mechanistically, MAD1 deficiency in hepatocytes enhances mitochondrial function and promotes hepatocyte proliferation by suppressing TGF-β signaling. Our study reveals MAD1 as a novel suppressor of hepatocyte proliferation, which may provide a new therapeutic target for the recovery of liver function after liver transplant and partial hepatectomy.

Proline-rich acidic protein 1 upregulates mitotic arrest deficient 1 to promote cisplatin-resistance of colorectal carcinoma by restraining mitotic checkpoint complex assembly.

Background: The mechanism underlying cisplatin resistance in colorectal carcinoma (CRC) has not yet been elucidated. This study is aimed to illustrate the indispensable role of proline-rich acidic protein 1 (PRAP1) in cisplatin-resistant CRC. Methods: Cell viability and apoptosis were monitored using cell counting kit-8 and flow cytometry. Immunofluorescence and morphological analysis were used to determine mitotic arrest in cells. In vivo drug resistance was evaluated using a tumor xenograft assay. Results: PRAP1 was highly expressed in cisplatin-resistant CRC. PRAP1-upregulation in HCT-116 cells increased chemoresistance to cisplatin, whereas RNAi-mediated knockdown of PRAP1 sensitized cisplatin-resistant HCT-116 cells (HCT-116/DDP) to cisplatin. PRAP1-upregulation in HCT-116 cells hindered mitotic arrest and the formation of mitotic checkpoint complexes (MCC), followed by an increase in multidrug-resistant proteins such as p-glycoprotein 1 and multidrug resistance-associated protein 1, while PRAP1-knockdown in HCT-116/DDP cells partly restored colcemid-induced mitotic arrest and MCC assembly, resulting in decreased multidrug-resistant protein levels. PRAP1 downregulation-mediated sensitization to cisplatin in HCT-116/DDP cells was abolished by the inhibition of mitotic kinase activity by limiting MCC assembly. Additionally, PRAP1-upregulation increased cisplatin-resistance in CRC in vivo. Mechanistically, PRAP1 increased the expression of mitotic arrest deficient 1 (MAD1), that competitively binds to mitotic arrest deficient 2 (MAD2) in cisplatin-resistant CRC cells, leading to failed assembly of MCC and subsequent chemotherapy resistance. Conclusion: PRAP1-overexpression caused cisplatin resistance in CRC. Possibly, PRAP1 induced an increase in MAD1, which competitively interacted with MAD2 and subsequently restrained the formation of MCC, resulting in CRC cells escape from the supervision of MCC and chemotherapy resistance.

Schizophrenia-associated Mitotic Arrest Deficient-1 (MAD1) regulates the polarity of migrating neurons in the developing neocortex

Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.

Mad1 Upregulation in Breast Cancer: Causes & Consequences

Mitotic arrest deficient-1 (Mad1), an essential component of the mitotic checkpoint, is upregulated in 20% of breast cancer patients. Upregulation of Mad1 causes chromosome missegregation during mitosis, a hallmark of cancer, as well as destabilization of the p53 tumor suppressor. Upregulation of Mad1 is sufficient to promote orthotopic tumor growth in immunocompromised animals. Patients with high levels of Mad1 mRNA expression have a poorer prognosis than patients with intermediate or low levels of Mad1. Unlike many core kinetochore proteins, Mad1 is not transcriptionally regulated by the transcription factor FoxM1. Thus, the mechanism for upregulating Mad1 in cancer, as well as the functional consequences, remain unknown. Bioinformatics analysis identified Histone Deacetylase 1 (HDAC1) as a likely cofactor involved in Mad1 transcriptional regulation. Consistent with this, HDAC inhibition with Trichostatin A (TSA) or Valproic Acid (VPA) increases Mad1 mRNA and protein levels 6- to 10-fold in multiple breast cancer cell lines. HDAC inhibition also induces Mad1 nuclear puncta, a localization pattern seen in primary breast cancer and breast cancer cell lines following Mad1 upregulation. Genetic approaches are currently being used to validate that HDAC1 negatively regulates Mad1 expression. To determine the consequences of Mad1 upregulation, we have generated a tetracycline (tet)-inducible Mad1 mouse model by inserting a tet responsive promoter and HA tag before the first coding exon of the Mad1 gene. Doubly heterozygous mice containing one allele of tet-inducible Mad1 and one allele of a ubiquitously expressed tet-transactivator (rtTA-M2) show inducible expression of HA-Mad1 following one week or one month of exposure to the tet analog doxycycline. Ongoing experiments will define the impact of Mad1 upregulation on mitotic fidelity and p53 levels and determine whether upregulation of Mad1 is sufficient to induce tumorigenesis in an immunocompetent setting.

Direct interactions of mitotic arrest deficient 1 (MAD1) domains with each other and MAD2 conformers are required for mitotic checkpoint signaling

As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset even when a single kinetochore is unattached to mitotic spindle microtubules. The key signal amplification reaction for the checkpoint is the conformational conversion of "open" mitotic arrest deficient 2 (O-MAD2) into "closed" MAD2 (C-MAD2). The reaction has been suggested to be catalyzed by an unusual catalyst, a MAD1:C-MAD2 tetramer, but how the catalysis is executed and regulated remains elusive. Here, we report that in addition to the well-characterized middle region of MAD1 containing the MAD2-interaction motif (MIM), both N- and C-terminal domains (NTD and CTD) of MAD1 also contribute to mitotic checkpoint signaling. Unlike the MIM, which stably associated only with C-MAD2, the NTD and CTD in MAD1 surprisingly bound both O- and C-MAD2, suggesting that these two domains interact with both substrates and products of the O-to-C conversion. MAD1NTD and MAD1CTD also interacted with each other and with the MPS1 protein kinase, which phosphorylated both NTD and CTD. This phosphorylation decreased the NTD:CTD interaction and also CTD's interaction with MPS1. Of note, mutating the phosphorylation sites in the MAD1CTD, including Thr-716, compromised MAD2 binding and the checkpoint responses. We further noted that Ser-610 and Tyr-634 also contribute to the mitotic checkpoint signaling. Our results have uncovered that the MAD1NTD and MAD1CTD directly interact with each other and with MAD2 conformers and are regulated by MPS1 kinase, providing critical insights into mitotic checkpoint signaling.

Proline‐rich acidic protein 1 (PRAP1) is a novel interacting partner of MAD1 and has a suppressive role in mitotic checkpoint signalling in hepatocellular carcinoma

Loss of mitotic checkpoint of cells contributes to chromosomal instability and leads to carcinogenesis. Mitotic arrest deficient 1 (MAD1) is a key component in mitotic checkpoint signalling. In this study, we identified a novel MAD1 interacting partner, proline-rich acidic protein 1 (PRAP1), using yeast-two hybrid screening, and investigated its role in mitotic checkpoint signalling in hepatocellular carcinoma (HCC). We demonstrated the physical interaction of PRAP1 with MAD1 and of PRAP1 with MAD1 isoform MAD1β, using a co-immunoprecipitation assay. Moreover, stable expression of PRAP1 in mitotic checkpoint-competent HCC cells, BEL-7402 and SMMC-7721, induced impairment of the mitotic checkpoint (p < 0.01), formation of chromosome bridges (p < 0.01) and aberrant chromosome numbers (p < 0.001). Interestingly, ectopic expression PRAP1 in HCC cells led to significant under-expression of MAD1. In human HCC tumours, 40.4% (23/57) of HCCs showed under-expression of PRAP1 protein as compared with their corresponding non-tumorous livers; up-regulation of MAD1 protein was significantly associated with down-regulation of PRAP1 (p = 0.030). Our data revealed that PRAP1 is a protein interacting partner of MAD1 and that PRAP1 is able to down-regulate MAD1 and suppress mitotic checkpoint signalling in HCC.

Abstract 572: Proline-rich acidic protein 1 (PRAP1), a novel mitotic arrest deficient 1 (MAD1) interacting partner, suppresses mitotic checkpoint signaling in hepatocellular carcinoma.

Abstract DNA aneuploidy or chromosomal instability is commonly observed in major types of human cancers, and one of the causes for chromosomal instability is defective mitotic checkpoint signaling in cells. Loss of mitotic checkpoint contributes to chromosomal instability, leading to carcinogenesis. The mitotic checkpoint monitors the status of kinetochore-microtubule attachment and delays the onset of anaphase until all kinetochores have been attached with microtubules. Mitotic arrest deficient 1 (MAD1) is key component in mitotic checkpoint signaling. We previously identified a novel isoform of MAD1, which we named MAD1β (Sze et al. Cancer Res 2008;68:9194-9201). In this study, using yeast-two hybrid screening, we have identified a novel MAD1 interacting partner, proline-rich acidic protein 1 (PRAP1). The function of PRAP1 is largely unknown. Hence, we investigated its role in mitotic checkpoint signaling in hepatocellular carcinoma (HCC). We demonstrated the physical interaction between PRAP1, MAD1 and MAD1β in yeast-two hybrid screening and with co-immunoprecipitation assay. Both MAD1 and MAD1β proteins were co-immunoprecipitated with PRAP1. Moreover, we stably expressed PRAP1 in two HCC cell lines, BEL-7402 and SMMC-7721, which have competent mitotic checkpoints. We observed that stable expression of PRAP1 induced impairment in mitotic checkpoint (P &amp;lt; 0.01). After treatment with either colcemid or nocodazole, the PRAP1-stably expressing BEL-7402 and SMMC-7721 cells showed significantly lower mitotic indices as compared with the corresponding vector control cells. Ectopic expression of PRAP1 also resulted in chromosome bridge formation (P&amp;lt; 0.01) and aberrant chromosome numbers (P &amp;lt; 0.001) in these cell lines. All these cellular changes contributed to chromosomal instability. Interestingly, ectopic expression PRAP1 led to significant underexpression of MAD1 but not MAD1β in HCC cells. Taken altogether, our data indicate that PRAP1 is an interacting partner of MAD1 and may have functions of degrading MAD1 and in turn suppressing mitotic checkpoint signaling in HCC. (This study was partly funded by a Hong Kong Research Grants Council GRF grant [HKU 772608]) Citation Format: Karen MF Sze, Glanice K. Chu, Queenie HY Mak, Irene OL Ng. Proline-rich acidic protein 1 (PRAP1), a novel mitotic arrest deficient 1 (MAD1) interacting partner, suppresses mitotic checkpoint signaling in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2013-572

The MAD1 1673 G→A polymorphism alters the function of the mitotic spindle assembly checkpoint and is associated with a worse response to induction chemotherapy and sensitivity to treatment in patients with advanced epithelial ovarian cancer

Mitotic arrest deficient 1 (MAD1), a protein of the mitotic spindle assembly checkpoint (SAC), recognizes MAD2 through two leucine zippers, transporting and activating MAD2, which promotes a metaphase arrest signal. A single nucleotide polymorphism of MAD1 was found to affect the SAC function that could be involved in a poor response to therapeutic agents that alter the dynamics of microtubules. The aim of this study was to examine the relationship of the polymorphism MAD1 1673 G → A (rs1801368) with the efficiency of the SAC and the generation of aneuploidies and with the therapeutic response of patients with ovarian cancer. The polymorphism was evaluated in 144 healthy individuals and 91 patients. Mitotic arrest and the presence of errors in segregation were analyzed in cultured human lymphocytes treated with nocodazole and paclitaxel. Errors in segregation were also evaluated in 27 biopsies of patients. Allele frequencies in healthy individuals were G: 50%, A: 50%, whereas in the patients they were G: 38%, A: 62% (P<0.05). The percentage of cells with mitotic arrest was higher among GG cells (P<0.05). The frequency of micronuclei and nondisjunction events increased in AA cells (P<0.05). Tumors from polymorphic patients had a higher percentage of aneuploid cells (P<0.05). The GG patients showed a higher biochemical response, optimal cytoreduction, and sensitivity to the treatment. There were no differences in progression-free or overall survival between both groups. The polymorphism MAD1 1673 G → A affects SAC functionality, increasing the frequency of aneuploid cells. This polymorphism modifies the response to agents that alter the dynamics of microtubules in patients with ovarian cancer.

Nuclear Factor κB–Mediated Transactivation of Telomerase Prevents Intimal Smooth Muscle Cell From Replicative Senescence During Vascular Repair

To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process by investigating expression and function of the catalytic telomerase reverse transcriptase (TERT) subunit after vascular injury. Functional telomerase is essential to the replicative longevity of vascular cells. We found that TERT was de novo activated in the intima of injured arteries, involving activation of the nuclear factor κB pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor α resulted in increased TERT activity. This depends on the activation of c-Myc signaling because mutation of the E-box in the promoter or overexpression of mitotic arrest deficient 1 (MAD1), a c-Myc competitor, abrogated the transcriptional activity. Inhibition of nuclear factor κB in both intimal SMCs and the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence after vascular injury. These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor κB and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.

Role of a Novel Splice Variant of Mitotic Arrest Deficient 1 (MAD1), MAD1β, in Mitotic Checkpoint Control in Liver Cancer

Loss of mitotic checkpoint contributes to chromosomal instability, leading to carcinogenesis. In this study, we identified a novel splicing variant of mitotic arrest deficient 1 (MAD1), designated MAD1beta, and investigated its role in mitotic checkpoint control in hepatocellular carcinoma (HCC). The expression levels of human MAD1beta were examined in hepatoma cell lines and human HCC samples. The functional roles of MAD1beta in relation to the mitotic checkpoint control, chromosomal instability, and binding with MAD2 were assessed in hepatoma cell lines. On sequencing, MAD1beta was found to have deletion of exon 4. It was expressed at both mRNA and protein levels in the nine hepatoma cell lines tested and was overexpressed in 12 of 50 (24%) human HCCs. MAD1beta localized in the cytoplasm, whereas MAD1alpha was found in the nucleus. This cytoplasmic localization of MAD1beta was due to the absence of a nuclear localization signal in MAD1alpha. In addition, MAD1beta was found to physically interact with MAD2 and sequester it in the cytoplasm. Furthermore, expression of MAD1beta induced mitotic checkpoint impairment, chromosome bridge formation, and aberrant chromosome numbers via binding with MAD2. Our data suggest that the novel splicing variant MAD1beta may have functions different from those of MAD1alpha and may play opposing roles to MAD1alpha in mitotic checkpoint control in hepatocarcinogenesis.

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein.

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.