Overexpression Of Mad2 Research Articles

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

Mad2 Induced Aneuploidy Contributes to Eml4-Alk Driven Lung Cancer by Generating an Immunosuppressive Environment

Simple SummaryIn tissue homeostasis, aneuploid cells have been suggested to be recognized and eliminated by immune cells. However, these initiating cancer cells can evade the immune system and ultimately derivate a tumor. To better understand how aneuploidy might contribute to tumor initiation and/or progression, we used two lung cancer models: one in which cancer cells are aneuploid and the surrounding normal epithelial cells are diploid, and a second one in which both tumor and normal cells are aneuploid. We show that aneuploid cells surrounding the tumor generate an immunosuppressive environment that contributes to lung cancer initiation.Aneuploidy, an imbalance number of chromosomes, is frequently observed in lung cancer and inversely correlates with patient survival. Paradoxically, an aneuploid karyotype has detrimental consequences on cellular fitness, and it has been proposed that aneuploid cells, at least in vitro, generate signals for their own elimination by NK cells. However, how aneuploidy affects tumor progression as well as the interplay between aneuploid tumor cells and the tumor microenvironment is still unclear. We generated a new mouse model in which overexpression of Mad2 was almost entirely restricted to normal epithelial cells of the lung, and combined it with an oncogenic Eml4-Alk chromosome inversion. This combination resulted in a higher tumor burden and an increased number of tumor nodules compared to control Eml4-Alk mice alone. The FISH analysis detected significant differences in the aneuploidy levels in the non-tumor regions of Eml4-Alk+Mad2 compared to Eml4-Alk alone, although both tumor groups presented similar levels of aneuploidy. We further show that aneuploid cells in the non-tumor areas adjacent to lung tumors recruit immune cells, such as tumor-associated macrophages. In fact, these areas presented an increase in alveolar macrophages, neutrophils, decreased cytotoxic CD8+ T cells, and IFN-γ, suggesting that aneuploid cells in the surrounding tumor areas create an immunosuppressive signature that might contribute to lung tumor initiation and progression.

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.

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.

Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

The mitotic checkpoint ensures proper segregation of chromosomes by delaying anaphase until all kinetochores are bound to microtubules. This inhibitory signal is composed of a complex containing Mad2, which inhibits anaphase progression. The complex can be disassembled by p31comet and TRIP13; however, TRIP13 knockdown has been shown to cause only a mild mitotic delay. Overexpression of checkpoint genes, as well as TRIP13, is correlated with chromosomal instability (CIN) in cancer, but the initialeffects of Mad2 overexpression are prolongedmitosis and decreased proliferation. Here, we showthat TRIP13 overexpression significantly reduced, and TRIP13 reduction significantly exacerbated, the mitotic delay associated with Mad2 overexpression, but not that induced by microtubule depolymerization. The combination of Mad2 overexpression and TRIP13 loss reduced the ability of checkpoint complexes to disassemble and significantly inhibited the proliferation of cells in culture and tumor xenografts. These results identify an unexpected dependency on TRIP13 in cells overexpressing Mad2.

Negative Selection and Chromosome Instability Induced by Mad2 Overexpression Delay Breast Cancer but Facilitate Oncogene-Independent Outgrowth.

SummaryChromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with KrasG12D or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.

Mad2 overexpression is associated with high cell proliferation and reduced disease-free survival in primary gastrointestinal diffuse large B-cell lymphoma

Objectives: Primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL) is a rare hematological malignancy with limited results on carcinogenesis and clinical characteristics. The aims of the current study were to examine mitotic arrest deficiency protein 2 (Mad2) expressions in PGI-DLBCL, and assess its association with Ki-67 expression, Helicobacter pylori (H. pylori) infection, BCL-6 gene rearrangement, and clinicopathological variables.Methods: Cancer tissues from 38 PGI-DLBCL patients were examined for Mad2, Ki-67, and H. pylori expression by immunohistochemistry, using normal gastrointestinal tissues and nodal DLBCL as controls. BCL-6 gene translocation was analyzed by fluorescence in situ hybridization (FISH), and Mad2 expression status was evaluated along with clinicopathological characteristics.Results: Mad2 expression was increased in PGI-DLBCL patients when compared with controls. The expression of Mad2 was 51.55 ± 22.88% in PGI-DLBCL, which was higher than reactive lymph node (28.77 ± 10.89%) and lymphoid nodule in normal gastrointestinal tissue (26.41 ± 11.30%) (P = 0.002), while it was comparable to nodal DLBCL (57.23 ± 20.79%) (P = 0.358). Mad2 overexpression had a positive correlation with Ki-67 proliferation index (r = 0.55, P = 0.01) in PGI-DLBCL, and patients with BCL-6 gene rearrangement had lower Mad2 expression (P = 0.032) than patients with intact BCL-6, while no relation was found between Mad2 expression and H. pylori infection. PGI-DLBCL patients with higher Mad2 expression had lower estimated disease-free survival (DFS) (17.10% vs. 53.00%) (P = 0.049). However, no correlation was found between Mad2 expression levels and overall survival (OS) (P = 0.443).Conclusions: Aberrant Mad2 expression was associated with cell proliferation and genetic instability, which may contribute to the carcinogenesis of PGI-DLBCL. Mad2 overexpression indicated a poor DFS and may be a potential biomarker for estimating prognosis for PGI-DLBCL patients.

Clinicopathologic significance of BubR1 and Mad2 overexpression in oral cancer.

BubR1 and Mad2 are central components of the mitotic checkpoint complex that inhibits anaphase onset until all chromosomes are correctly aligned at the metaphase plate. We propose to analyse the combined expression of BubR1 and Mad2 and assess its significance to oral squamous cell carcinoma (OSCC) diagnosis and prognosis. BubR1 and Mad2 expression was assessed by real-time PCR in OSCC cell lines and in normal human oral keratinocytes, and by immunohistochemistry in 65 patients with OSCC. The results were compared regarding clinicopathological parameters, proliferative activity and survival. BubR1 and Mad2 transcripts were overexpressed in OSCC cell lines which also exhibited attenuated spindle assembly checkpoint activity. BubR1 and Mad2 were also overexpressed in patients with OSCC. BubR1 expression was associated with advanced stages and larger tumour size in univariate analysis, and with shorter overall survival both in univariate and multivariate analysis. Mad2 overexpression was associated with that of BubR1 and, importantly, high expression of Mad2 and BubR1 was associated with increased cellular proliferation. Our data propose a role for BubR1 and Mad2 in OSCC cellular proliferation, progression and prognosis.

Structure-biological function relationship extended to mitotic arrest-deficient 2-like protein Mad2 native and mutants-new opportunity for genetic disorder control.

Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53–0.65) and fitted correlation r2 (0.82–0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN.

The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B.

The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF(-/-) MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF(-/-) cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF(-/-) MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.

Withaferin A modulates the Spindle Assembly Checkpoint by degradation of Mad2–Cdc20 complex in colorectal cancer cell lines

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability.

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect. Because the spindle checkpoint pathway is highly conserved between yeast and humans, we performed a synthetic genetic array analysis in yeast, which revealed that Mad2 overexpression induced lethality in 13 gene deletions. Among the human homologs of candidate genes, knockdown of PPP2R1A, a gene encoding a constant regulatory subunit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 phosphorylation and suppressed Mad2 protein levels. Depletion of PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa cells but not of unphosphorylated Mad2 mutant-overexpressing cells, suggesting that the lethality induced by PP2A depletion in Mad2-overexpressing cells is dependent on Mad2 phosphorylation. Also, the PP2A inhibitor cantharidin induced Mad2 phosphorylation and inhibited the growth of Mad2-overexpressing cancer cells. Aurora B knockdown inhibited Mad2 phosphorylation in mitosis, resulting in the blocking of PPP2R1A inhibition-induced cell death. Taken together, our results strongly suggest that PP2A is a good therapeutic target in Mad2-overexpressing tumors.

Upregulation of Mad2 facilitates in vivo and in vitro osteosarcoma progression

It has been reported that overexpression of Mad2 in transgenic mice leads to a wide variety of tumors, and Mad2 overexpression causes lung tumor relapse after oncogene withdrawal. In a previous study we demonstrated that Mad2 is abnormally upregulated in human osteosarcoma, however, the underlying mechanisms remain unknown. In this study, we found that transient Mad2 overexpression is sufficient to cause early dyscrasia and decreased survival in a xenotransplantation osteosarcoma mouse model, and Mad2 overexpression is associated with increased invasiveness and pulmonary metastasis. We also found that upregulation of Mad2 was accompanied by enhanced capability to self-renew. Our data validate the correlation between upregulation of Mad2 and osteosarcoma advancement, and that the underlying mechanisms involve the increase of invasiveness and cancer stem cell properties.

Checkpoint-Independent Stabilization of Kinetochore-Microtubule Attachments by Mad2 in Human Cells

Faithful chromosome segregation is required for cell and organism viability and relies on both the mitotic checkpoint and the machinery that corrects kinetochore-microtubule (k-MT) attachment errors. Most solid tumors have aneuploid karyotypes and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). Mad2 is essential for mitotic checkpoint function and is frequently overexpressed in human tumors that are CIN. For unknown reasons, cells overexpressing Mad2 display high rates of lagging chromosomes. Here, we explore this phenomenon and show that k-MT attachments are hyperstabilized by Mad2 overexpression and that this undermines the efficiency of correction of k-MT attachment errors. Mad2 affects k-MT attachment stability independently of the mitotic checkpoint because k-MT attachments are unaltered upon Mad1 depletion and Mad2 overexpression hyperstabilizes k-MT attachments in Mad1-deficient cells. Mad2 mediates these effects with Cdc20 by altering the centromeric localization and activity of Aurora B kinase, a known regulator of k-MT attachment stability. These data reveal a new function for Mad2 to stabilize k-MT attachments independent of the checkpoint and explain why Mad2 overexpression increases chromosome missegregation to cause chromosomal instability in human tumors.

CDK-Dependent Potentiation of MPS1 Kinase Activity Is Essential to the Mitotic Checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Mad2 Is a Critical Mediator of the Chromosome Instability Observed upon Rb and p53 Pathway Inhibition

Multiple mechanisms have been proposed to explain how Rb and p53 tumor suppressor loss lead to chromosome instability (CIN). It was recently shown that Rb pathway inhibition causes overexpression of the mitotic checkpoint gene Mad2, but whether Mad2 overexpression is required to generate CIN in this context is unknown. Here, we show that CIN in cultured cells lacking Rb family proteins requires Mad2 upregulation and that this upregulation is also necessary for CIN and tumor progression invivo. Mad2 is also repressed by p53 and its upregulation is required for CIN in a p53 mutant tumor model. These results demonstrate that Mad2 overexpression is a critical mediator of the CIN observed upon inactivation of two major tumor suppressor pathways.

Abstract LB-192: DNA damage checkpoints control spindle assembly checkpoint by regulating Mad2

Abstract DNA damage checkpoints coordinate with the spindle assembly checkpoint (SAC) to maintain genome integrity. In yeast Saccharomyces cerevisiae, ATM/ATR-Chk2/Chk1 checkpoint enhances SAC through phosphorylation and stabilization of securin by Chk1 and inhibition of APCCDC20 by Chk2 in response to DNA damage. The control of SAC by DNA damage checkpoints in mammalian cells is unclear. Here we report that DNA damage checkpoint signals to SAC by regulating Mad2 in human rhabdomyosarcoma (RMS) cells. In vitro, treatment of RMS Rh30 cells with DNA strand crosslinkers melphalan, cisplatin or mitomycin C for 5 hours resulted in elevation of Mad2 protein, which was companied with ATM/ATR checkpoint activation as shown by increase of ATM-S1981, Chk2-T68 and Chk1-S345. It has been shown that Mad2 is controlled by the Rb-E2F1 pathway and overexpression of Mad2 leads to genome instability and tumorigenesis. Consistently, treatment of Rh30 cells with inhibitors of either mTOR, Akt or CDK1/2 lead to dephosphorylation of Rb and decrease of Mad2. However, we found that DNA damage-induced increase of Mad2 is independent on Rb-E2F1 pathway as treatment of Rh30 cells with DNA damage agents resulted in dephosphorylation of Rb, suggesting inhibition of the E2F1 pathway. Moreover, we found that Mad2 is controlled by ubiquitin-proteasome signaling as MG132 treatment increased Mad2 protein levels. In vivo, we determined the Mad2 levels in 50 pediatric xenograft tumor models (PPTP) and found that Mad2 is overexpressed in most RMS (7/8). Elevated Mad2 is associated with elevated spontaneous DNA damage response in RMS as demonstrated by relatively high levels of Chk2-T68 and Chk1-S345 phosphorylation. The deregulation of the Rb-E2F1 pathway seems not to be the cause of Mad2 overexpression in RMS as Rb-S780 phosphorylation is comparable to other PPTP tumors without Mad2 overexpression. Further, gene expression and copy number analysis found that chromosome 2p24, in which MYCN is located, is amplified in most alveolar RMS xenografts (58.3%). Consequently, both MYCN mRNA and protein are overexpressed in these 2p24 amplified RMS. It has been well documented that oncogene activation leads to DNA replication stress and consequently DNA damage response in most cancers especially late-stage and relapse cancers. Alveolar RMS is genomically unstable as shown by increased tetraploid cells. We thus hypothesize that chromosome 2p24 amplification results in overexpression of MYCN oncogene, which leads to replication stress and hence DNA damage, and this oncogene induced DNA damage may lead to overexpression of Mad2 and consequently chromosome instability in RMS. Taken together, we speculate that DNA damage response stabilizes Mad2 protein independently of the Rb-E2F1 pathway in vitro. In vivo, oncogene amplification leading to an enhanced DNA damage response may stabilize Mad2 overexpression, potentially leading to chromosome instability. Supported by CA77776. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2011-LB-192

Mad2-induced chromosome instability leads to lung tumour relapse after oncogene withdrawal

Inhibition of an initiating oncogene often leads to extensive tumor cell death, a phenomenon known as oncogene addiction1. This has led to the search for compounds that specifically target and inhibit oncogenes as anti-cancer agents. Whether chromosomal instability (CIN) generated as a result of deregulation of the mitotic checkpoint pathway2,3, a frequent characteristic of solid tumors, has any effect on oncogene addiction, however, has not been explored systematically. We show here that induction of chromosome instability by overexpression of the mitotic checkpoint gene Mad2 does not affect the regression of Kras driven lung tumors upon Kras inhibition. However, tumors that experience transient Mad2 overexpression and consequent chromosome instability recur at dramatically elevated rates. The recurrent tumors are highly aneuploid and have varied activation of pro-proliferative pathways. Thus, early CIN may be responsible for tumor relapse after seemingly effective anti-cancer treatments.

BUBR1 and MAD2: Novel Markers for Predicting Benefit from Adjuvant Anthracyclines?

Abstract Background: We previously demonstrated the importance of duplication of CEP17 (C17) in predicting sensitivity to anthracyclines in two retrospective adjuvant trials (BR9601 and MA.5). Spindle checkpoint failure, and resultant chromosome instability (CIN), could be a mechanism for this observation. BubR1 and MAD2 are key components of the spindle assembly checkpoint which prevent progression to anaphase. Overexpression of BUBR1 or a reduction in MAD2 is associated with CIN and polysomy. Unlike MAD2, BubR1 competitively binds, phosphorylates and stabilises p53 during mitosis, at a site overlapping that of MDM2. Therefore, BubR1 is implicated in both spindle assembly and DNA damage checkpoints. We investigated BubR1 and MAD2 as predictive markers of anthracycline sensitivity in breast cancer.Methods: We evaluated cytoplasmic BubR1 and MAD2 protein expression in 321 tumours from patients in the BR9601 trial, which compared E-CMF with CMF as adjuvant therapy for breast cancer. Overall survival (OS) and relapse-free survival (RFS) were estimated using Kaplan-Meier curves.Results: BubR1 and MAD2 overexpression were detected in 49.2% and 25.1% of tumors, respectively. Both MAD2 and BubR1 overexpression were associated with ER negativity (p&amp;lt;0.0001, p=0.013) and high grade (p&amp;lt;0.0001, p=0.029). BubR1 overexpression was associated with C17 (p=0.0003) and high proliferation (Ki67, p&amp;lt;0.0001); similar trends were noted for MAD2. BubR1 overexpression was associated with reduced RFS (HR: 1.58, 95%C.I. 1.06-2.37, p=0.024) and OS (HR: 1.831 95%C.I. 1.161-2.890), whereas there was no clear association with outcome for MAD2.Interaction tests revealed no significant evidence that the benefit of the addition of epirubicin to CMF was confined to those tumours with either high levels of BubR1 or low levels of MAD2 (BubR1 HR: 1.70 95%C.I. 0.74-3.87 and MAD2 HR: 0.573 95%C.I. 0.143-2.296). However, combining overexpression of BubR1 with low expression of MAD2 selects for 33.0% (98/297) of patients and is associated overall with a poorer RFS (HR: 0.602 95%C.I. 0.402-0.902, p=0.014) but an increased RFS benefit from the addition of anthracycline to CMF (HR: 0.409 95%C.I. 0.208-0.803) than those with any other combination of BubR1 and MAD2 expression (HR: 0.718 95%C.I. 0.423-1.216). Similar results were noted with OS. However, neither treatment by marker interaction reached significance (RFS HR: 1.73 95%C.I. 0.74-1.08, p=0.208; OS HR 1.820 95%C.I. 0.707-4.686, p=0.215).Conclusion: BubR1 overexpression is associated with an aggressive phenotype and decreased RFS and OS in BR9601 patients. Tumours with BubR1 overexpression and/or low MAD2 expression showed a potential increased sensitivity to anthracycline therapy compared to those with low BubR1 levels and/or high MAD2 expression. Small trials (BR9601) are useful for hypothesis generating; further work in larger patient cohorts such as MA.5 or NEAT is warranted. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2124.