Mad2 Expression Research Articles

Manipulation of the Myc Interactome to Enhance Nerve Regeneration in a Murine Model.

This study was undertaken to explore manipulation of the Myc protein interactome, members of an oncogene group, in enhancing the intrinsic growth of injured peripheral adult postmitotic neurons and the nerves they supply. New approaches to enhance adult neuron growth properties are a key strategy in improving nerve regeneration. Expression and impact of Myc interactome members c-Myc, N-Myc, Mad1, and Max were evaluated within naive and "preconditioned" adult sensory neurons and Schwann cells (SCs), using siRNA and transfection of CRISPR/Cas9 or luciferase reporter in vitro. Morphological, behavioral, and electrophysiological indices of nerve regeneration were analyzed in vivo. c-Myc, N-Myc, Max, and Mad were expressed in adult sensory neurons and in partnering SCs. In vitro knockdown (KD) of either Mad1 or Max, competitive inhibitors of Myc, unleashed heightened neurite outgrowth in both naive uninjured or preconditioned adult neurons. In contrast, KD or inhibition of both isoforms of Myc was required to suppress growth. In SCs, Mad1 KD not only enhanced migratory behavior but also conditioned increased outgrowth in separately cultured adult sensory neurons. In vivo, local Mad1 KD improved electrophysiological, behavioral, and structural indices of nerve regeneration out to 60 days of follow-up. Members of the Myc interactome, specifically Mad1, are novel targets for improving nerve regeneration. Unleashing of Myc growth signaling through Mad1 KD enhances the regrowth of both peripheral neurons and SCs to facilitate better regrowth of nerves. ANN NEUROL 2024;96:216-230.

Regulatory effects of miR-19a on MAD2 expression and tumorigenesis in gastric cancer

Regulatory effects of miR-19a on MAD2 expression and tumorigenesis in gastric cancer

Anti-proliferative, pro-apototic and anti-migratory properties of HDAC inhibitor PXD-101 on osteosarcoma cell lines

The therapeutic strategies for osteosarcoma involve both surgical approach and chemotherapy, but the identification of new therapeutic targets is particularly necessary in patients with local chemo-resistance, recurrence and lung metastases. The role of epigenetic regulation in osteosarcoma is largely unknown. Thus, in this study we disclosed the effects of histone deacetylase inhibitor drug PXD-101 on human osteosarcoma (OS) cell lines with different aggressiveness, including Saos-2, HOS and 143B cell lines. XTT assays revealed that treatment of Saos-2, HOS and 143B cells with PXD-101 decreased cell viability in a concentration-dependent manner. Fluorescence-activated cell sorting (FACS) analysis showed that PXD-101 inhibited proliferation and induced cell apoptosis. Wound healing assay indicated that PXD-101 inhibited migration of osteosarcoma cells. Real-Time RT-qPCR and protein analysis highlighted reduced expression of Runx2, Osterix and Mad2, probably due to Cyclin B1 inhibition by PXD-101 treatment. To our knowledge, this is the first study that characterized the anti-tumoral effect of PXD-101 in OS cells, suggesting a potential new therapeutic approach in osteosarcoma patients.

SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

BackgroundShugoshin-like protein 2 (SGOL2) is a centromeric protein that ensures the correct and orderly process of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis. Here, we examined the potential role of SGOL2 in cancers, especially in hepatocellular carcinoma (HCC).MethodsOne hundred ninety-nine normal adjacent tissues and 202 HCC samples were collected in this study. Human HCC cells (SK-HEP-1 and HEP-3B) were employed in the present study. Immunohistochemistry, immunofluorescence, western blot, Co-Immunoprecipitation technique, and bioinformatic analysis were utilized to assess the role of SGOL2 in HCC development process.ResultsOverexpression of SGOL2 predicted an unfavorable prognosis in HCC by The Cancer Genome Atlas database (TCGA), which were further validated in our two independent cohorts. Next, 47 differentially expressed genes positively related to both SGOL2 and MAD2 were identified to be associated with the cell cycle. Subsequently, we demonstrated that SGOL2 downregulation suppressed the malignant activities of HCC in vitro and in vivo. Further investigation showed that SGOL2 promoted tumor proliferation by regulating MAD2-induced cell-cycle dysregulation, which could be reversed by the MAD2 inhibitor M2I-1. Consistently, MAD2 upregulation reversed the knockdown effects of SGOL2-shRNA in HCC. Moreover, we demonstrated that SGOL2 regulated MAD2 expression level by forming a SGOL2-MAD2 complex, which led to cell cycle dysreuglation of HCC cells.ConclusionSGOL2 acts as an oncogene in HCC cells by regulating MAD2 and then dysregulating the cell cycle, providing a potential therapeutic target in HCC.

High-dose metformin induces a low-glucose dependent genotoxic stress

Epidemiological studies have demonstrated that metformin (a cornerstone of diabetes treatment) has anticancer activity, but the underlying mechanism remains elusive. We aimed to investigate whether metformin elicits anticancer activity via increasing genotoxic stress, a state of increased genome damage that becomes tumor-suppressing if it goes beyond an intolerable threshold. We found that metformin (1–16 mM) suppressed proliferation and colony formation in a panel of cancer cell lines (HeLa, A375, A549 and QGY). Metformin induced a dose-dependent increase of genotoxic stress (including micronucleus, nucleoplasmic bridge and nuclear bud) and the increase of genotoxic stress correlated well with metformin's anticancer potential. Metformin deregulated the expression of BUBR1 and MAD2, two core genes of spindle assembly checkpoint (SAC) that surveillances chromosome segregation. Metformin had weakened antiproliferative effect and a corresponding attenuated genotoxic effect in HeLa cells cultured in high glucose (16 mg/ml). Meanwhile, metformin significantly increased genotoxicity in non-cancer cells (NCM460 and HUVECs). Metformin became non-genotoxic to HUVECs in high-glucose (8 and 16 mg/ml) conditions and reduced the genotoxicity of high glucose. Overall, these results infer a new mechanism of high-dose metformin, whereby low-glucose dependent genotoxic stress derived from SAC dysfunction might mediate some of the anticancer effect of this drug.

Age-dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B.

A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore–microtubule attachments and recruit SAC regulator proteins. In mice, oocyte‐specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II‐arrested eggs and premature age‐related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.

PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

Dihydrotanshinone exerts antitumor effects and improves the effects of cisplatin in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer and is responsible for 20‑50% of thyroid cancer‑associated deaths. The absence of response to conventional treatments makes the search for novel therapeutics a clinical challenge. In the present study, the effects of 15,16‑dihydrotanshinoneI (DHT), a tanshinone extracted from Salviamiltiorrhiza Bunge (Danshen), which has previously been shown to possess anticancer activity, were examined in two human ATC cell lines. DHT significantly reduced cell viability, which was coupled with an increase in apoptosis. DHT administration also reduced the colony‑forming ability and proliferation of these cells in soft agar and downregulated the expression of epithelial‑to‑mesenchymal transition‑related genes. In addition, DHT significantly reduced MAD2 expression, a target of HuR with a relevant role in ATC. Finally, cotreatment with cisplatin and DHT has a greater effect on cell viability than each compound alone. In conclusion, to the best of our knowledge, the present study is the first to demonstrate that DHT exerts antitumor effects on ATC cells by reducing MAD2 expression levels. Moreover, a synergistic effect of DHT with cisplatin was shown. Further invivo studies are required to assess this phytochemical compound as a potential adjuvant for the treatment of ATC.

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.

Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

BackgroundAneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date.MethodsIn this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting.Resultsstatistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b.ConclusionThese results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

Expression of spindle assembly checkpoint proteins BubR1 and Mad2 expression as potential biomarkers of malignant transformation of oral leukoplakia: an observational cohort study.

Background The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia.Material and Methods We analysed the immunoexpression of BubR1, Mad2, Bub3, and Spindly proteins in 64 oral biopsies from 52 oral leukoplakias and 12 normal tissues. Univariate and multivariate analysis were performed to evaluate predictive factors for malignant transformation (MT).Results We observed that BubR1 and Mad2 were more highly expressed in high dysplasia grade lesions than in low grade or normal tissues (P<0.05). High expression of Spindly was significantly correlated with a high Ki-67 score (P=0.004). Six (11.5%) oral leukoplakias underwent malignant transformation. In univariate analysis, the binary dysplasia grade (high grade) (P<0.001) was associated with a higher risk of malignant transformation as well as high BubR1 (P<0.001) and high Mad2 (P=0.013) expression. In multivariate analysis, high expression of BubR1 and Mad2 when combined showed an increased risk for malignant transformation (P=0.013; HR of 4.6, 95% CI of 1.4-15.1).Conclusions Our findings reveal that BubR1 and Mad2 were associated with an increased risk for malignant transformation independently of histological grade and could be potential and useful predictive risk markers of malignant transformation in oral leukoplakias. Key words:BubR1, Mad2, Spindly, Bub3, Oral Leukoplakia, epithelial dysplasia, Oral squamous cell carcinoma.

The role of the MAD2-TLR4-MyD88 axis in paclitaxel resistance in ovarian cancer.

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.

Analysis of TET2 and EZH2 gene functions in chromosome instability in acute myeloid leukemia

TET2 and EZH2 play important roles in the epigenetic regulation in many cancers. However, their specific roles in acute myeloid leukemia (AML) pathogenesis remain unknown. Here, the expression, methylation or mutation of EZH2 and TET2 was determined and further correlated with the levels of the chromosome instability (CIN) genes MAD2 and CDC20. We down-regulated EZH2 and TET2 in AML cell lines and assessed the effect on CIN using fluorescence in situ hybridization (FISH). Our results showed that TET2, EZH2, MAD2 and CDC20 were aberrantly expressed in AML patients. The expression level of MAD2 or CDC20 was positively correlated with that of TET2 or EZH2. Hypermethylation of the TET2 gene down-regulated its transcription. Down-regulation of EZH2 or TET2 expression inhibited apoptosis, affected MAD2 and CDC20 expression, and promoted CIN in AML cells. Decitabine treatment restored TET2 methylation and EZH2 transcription and ameliorated CIN in AML. Therefore, TET2 and EZH2 play a tumor-inhibiting role in AML that affects CIN via MAD2 and CDC20.

Too MAD or not MAD enough: The duplicitous role of the spindle assembly checkpoint protein MAD2 in cancer.

MAD2 is an intriguing protein, which has been associated with poor survival in cancer. Depending on the organ-specific cancer, either high expression or low expression levels have been correlated with low survival rates in patients. MAD2 is also a marker of contradiction. The normal function of MAD2 is to accumulate at kinetochores and generate a wait signal preventing the cell from progressing to anaphase of the cell cycle until the spindle microtubules have correctly aligned with the kinetochores on each chromosome. This process ensures that sister chromatids segregate correctly into each new daughter cell upon cellular division. Thus, the correct function of MAD2 and this crucial cell cycle checkpoint, the spindle assembly checkpoint (SAC), is essential for faithful replicative cell division, the prevention of chromosomal abnormalities and the development of cancer. Surprisingly when MAD2 is supressed for example through siRNA, this results in the induction of cellular senescence or cell cycle arrest. This is an inherent contradiction as normally the dispersement of MAD2 would signal to a cell that they should proceed to anaphase as spindle microtubules have correctly aligned with each chromatid for cell division. In the inverse setting; a second contradiction, high MAD2 expression in cancer patients generally correlates with abnormal chromosome number. However, in normal cells high expression of MAD2 would limit this by generating a wait signal to prevent the cell from proceeding through the cell cycle. In this review article we aim to make sense of the MADness and review the current knowledge of MAD2 and its role in cancer.

Downregulation of Mad2 and BubR1 increase the malignant potential and nocodazole resistance by compromising spindle assembly checkpoint signaling pathway in cervical carcinogenesis.

To explore the involvement of Mad2 and BubR1 in cervical carcinogenesis. The expressions of Mad2 and BubR1 in tissues of high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and chronic cervicitis were analyzed immunohistochemistrily and compared with those of p16INK4A . PEGFP-Mad2 and pEGFP-BubR1 were transfected into SiHa cells to overexpress Mad2 and BubR1 and Si-RNAs to knockdown. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Migration and invasion capabilities were detected by Transwell. Propidium iodide staining with flow cytometry was used for cell cycle analysis and apoptosis was detected using Annexin V/7-AAD staining after nocodazole treatment. The expression of Mad2 was significantly lower in HSIL than those in chronic cervicitis and LSIL, however, the expression of BubR1 showed no significant differences. To detect HSIL in cervical lesions, Mad2 had a sensitivity of 88.44% and a specificity of 87.23%, Mad2 was less sensitive and more specific than p16INK4a . In SiHa cells, knockdown of Mad2 and BubR1 increased cell growth, reinforced invasion capacity and migration potency, inhibited apoptosis and decreased G2-phase distribution after nocodazole treatment. Oppositely, the overexpression strategies made cells show decreased malignant behaviors, raised apoptosis and increased G2-phase distribution. Mad2 negativity was specific to identify HSIL immunohistochemistrily. Downregulation of Mad2 and BubR1 increase the malignant behavior and nocodazole resistance of SiHa cells via causing spindle assembly checkpoint defect. This mechanism may contribute to cervical carcinogenesis and resistance to microtubule-targeting drugs.

Aurora B prevents aneuploidy via MAD2 during the first mitotic cleavage in oxidatively damaged embryos.

ObjectivesA high rate of chromosome aneuploidy is exhibited in in vitro fertilization (IVF)‐derived embryos. Our previous experiments suggested that reactive oxygen species (ROS) can activate Mad2, a key protein in the spindle assembly checkpoint (SAC), and delay the first mitotic, providing time to prevent the formation of embryonic aneuploidy. We aimed to determine whether mitotic kinase Aurora B was involved in the SAC function to prevent aneuploidy in IVF‐derived embryos.Materials and MethodsWe analysed aneuploidy formation and repair during embryo pre‐implantation via 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) staining and karyotype analysis. We assessed Aurora B activation by immunofluorescence and investigated the effect of Aurora B inhibition on embryo injury‐related variables, such as embryonic development, ROS levels, mitochondrial membrane potential and γH2AX‐positive expression.ResultsWe observed the expression and phosphorylation of Thr232 in Aurora B in oxidative stress‐induced zygotes. Moreover, inhibition of Aurora B caused chromosome mis‐segregation, abnormal spindle structures, abnormal chromosome number and reduced expression of Mad2 in IVF embryos. Our results suggest that Aurora B causes mitotic arrest and participates in SAC via Mad2 and H3S10P, which is required for self‐correction of aneuploidies.ConclusionsWe demonstrate here that oxidative stress–induced DNA damage triggers Aurora B‐mediated activation of SAC, which prevents aneuploidy at the first mitotic cleavage in early mouse IVF embryos.

Pign Suppresses CIN and Leukemia Progression Via Regulation of the Spindle Assembly Checkpoint

The spindle assembly checkpoint complex (SAC) is responsible for proper chromosomal segregation during mitosis. The SAC stalls mitotic exit until proper attachment of mitotic spindles to the chromosomes and bi-orientation of the chromosomes on the spindles are achieved. Dysregulation of the SAC may result in chromosomal instability (CIN) which is known to drive leukemia progression. We previously assessed the impact of phosphatidylinositol glycan anchor biosynthesis class N (PIGN) expression aberrations on leukemia progression and showed that PIGN expression aberrations were linked with CIN and leukemia transformation in high-risk myelodysplastic syndrome (MDS) patients. An in-depth understanding of the mechanistic basis of PIGN involvement in CIN and leukemic progression would have boundless therapeutic and diagnostic implications for patients. Thus, we investigated the mechanistic link between PIGN, CIN and the SAC.PIGN downregulation via RNAi and CRISPR/Cas9 as well as ectopic overexpression studies, co-immunoprecipitation, and confocal microscopy were employed to decipher the relationship between PIGN, CIN, and SAC signaling. Additionally, we tested whether the depletion of PIGN results in aberrant cell cycle signaling and defective chromosomal segregation using flow cytometry and mitotic index assays. We initially performed cell cycle synchronization experiments using myeloid and lymphoblastoid cell lines and examined PIGN expression at different stages of the cell cycle via Western blot analyses and RT-qPCR.Our results indicated that PIGN expression was cell cycle-regulated and PIGN loss significantly impacted the expression of SAC-related proteins. CRISPR/Cas9 mediated knockout of PIGN in CD34+ mononuclear cells derived from a healthy individual resulted in the suppression of MAD1 and MAD2. A similar observation was made in HEK293 PIGN CRISPR/Cas9 knockout cells. PIGN loss in the HEK293 cells resulted in MAD1, MAD2, and MPS1 suppression but led to BUBR1 upregulation. PIGN downregulation resulted in impaired mitotic checkpoint activation and consequently impacted mitotic exit. PIGN downregulation results in defective mitotic checkpoint signaling and mitotic exit with an accumulation of missegregation errors. Interestingly, ectopic overexpression of PIGN restored the MAD1 and MAD2 expression. Co-immunoprecipitation experiments and confocal analyses in cell cycle synchronized cells respectively revealed direct interactions and co-localization between PIGN and the SAC proteins MAD1, MAD2, as well as the mitotic kinase MPS1 thus unveiling a novel spatiotemporal regulatory mechanism. PIGN physically interacts with and regulates the SAC via MAD1, MAD2, MPS1 and BUBR1 during mitotic cell cycle progression. The co-purification of PIGN with some of these mitotic checkpoint proteins showed the direct role that PIGN may play in the regulation of mitotic checkpoint signaling. Thus, PIGN as a CIN suppressor may be crucial in the regulation of mitotic integrity via the SAC as part of maintaining genome stability.Despite the ubiquity of CIN in leukemia progression, there is still limited knowledge about the mechanism(s) involved. Also, since its discovery as a CIN suppressor, the molecular mechanism by which the loss of PIGN leads to CIN has until now remained elusive. However, this study revealed a novel mechanism in which PIGN may maintain genome stability via SAC regulation. Our findings open the possibility to study PIGN as a tumor suppressor because its loss significantly altered the expression of SAC-related proteins. Ultimately, PIGN modulation could be adopted as a therapeutic approach in leukemia treatment, more specifically in the averting leukemia progression in high-risk MDS patients. DisclosuresNo relevant conflicts of interest to declare.

MAD2 Combined with Mitotic Spindle Apparatus (MSA) and Anticentromere Antibody (ACA) for Diagnosis of Small Cell Lung Cancer (SCLC).

BackgroundMAD2 is the gene controlling mitosis. Many studies have assessed MAD2 in various types of carcinoma. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) are related mitotic antibodies, playing roles in autoimmune diseases and carcinomas, but the expression of MAD2, MSA, and ACA in SCLC is unclear.Material/MethodsWe enrolled 70 SCLC patients, 72 non-small cell lung cancer (NSCLC) patients, and 65 pulmonary nodule (PN) patients. MAD2 expression was measured through agarose electrophoresis and qt-PCR. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) were detected by indirect immunofluorescence (IIF).ResultsMAD2 was found both in SCLC and NSCLC. Interestingly, there was a significant difference found between SCLC and NSCLC using qt-PCR (P<0.05). The area under the ROC curve of MAD2 expression was 0.799, with medium diagnostic value. MAD2 expression was related to age, lymphatic metastasis, and survival time, but not with sex. The positivity for MSA and ACA by IIF assay were 37.20% and 34.00%, respectively, in the SCLC group, which were higher than in the NSCLC and pulmonary nodule groups (P<0.05). The kappa values of MSA and ACA with MAD2 expression were 0.73 and 0.65, respectively, with moderate consistency. Combining MAD2 with MSA and ACA enhanced the sensitivity and specificity for diagnosing SCLC.ConclusionsMAD2 expression was found to be involved in carcinogenesis and prognosis of SCLC. The combination of MAD2 with MSA and ACA is useful for early diagnosis and shows promise in treatment of SCLC.

Correction: Significance of MAD2 Expression to Mitotic Checkpoint Control in Ovarian Cancer Cells

Correction: Significance of MAD2 Expression to Mitotic Checkpoint Control in Ovarian Cancer Cells

PO-224 consequences of FOXP1-mediated transcriptional repression of the mitotic checkpoint gene MAD2 in breast cancer

IntroductionThe mitotic checkpoint, or spindle assembly checkpoint (SAC), is a safeguard mechanism that prevents chromosome missegregation during mitosis. It is well established that overexpression of the mitotic checkpoint component MAD2 leads to chromosome instability (CIN) and tumorigenesis. MAD2 expression is controlled by transcriptional activator and repressor E2Fs, as well as by cell cycle dependent element/cell cycle homology region (CDE/CHR) sequences in its promoter. However, precisely how MAD2 transcription is regulated has remained elusive. Focusing on breast cancer, we therefore aimed to identify novel MAD2 transcriptional regulators and to characterise how these might contribute to CIN and tumour formation.Material and methodsPotential MAD2 transcriptional regulators were identified using a DNA-protein pull-down assay and mass spectrometry. In-depth characterisation of protein-DNA interactions and transcriptional effects were studied using chromatin immunoprecipitation, promoter mutagenesis and reporter assays. Cell lines in which identified repressors were stably knocked-down by shRNAs were used to investigate changes in MAD2 protein levels, cell cycle progression and chromosome segregation fidelity. In silico analyses and immunohistochemistry on tissue microarrays were used to study associations between expression of the regulators and MAD2 at the mRNA and protein levels in breast cancer. Repressor expression levels were also assessed in the context of breast cancer patient prognosis and survival.Results and discussionsWe identified the forkhead box transcription factor FOXP1 as a main transcriptional repressor of MAD2 expression. FOXP1 reduces MAD2 promoter activity via direct binding to CDE/CHR elements. Furthermore, knockdown of FOXP1 expression increases the proportion of cells in G2/M phase. In addition, low FOXP1 expression strongly correlates with high MAD2 expression at the mRNA and protein levels, and with poor patient prognosis in breast cancer, especially triple-negative breast cancer.ConclusionWe identified FOXP1 as a novel transcriptional repressor of MAD2 expression. Our results suggest that reduced FOXP1 levels in breast cancer affect both MAD2 expression and cell cycle progression. This may promote CIN and tumour formation via upregulation of MAD2. Our results may have important implications for breast cancer diagnostics and, potentially, therapeutic targeting.