Defective Mitotic Checkpoint Research Articles

CCNF (Cyclin F) as a Candidate Gene for Familial Hodgkin Lymphoma: Additional Evidence for the Importance of Mitotic Checkpoint Defects in Tumorigenesis.

CCNF (Cyclin F) as a Candidate Gene for Familial Hodgkin Lymphoma: Additional Evidence for the Importance of Mitotic Checkpoint Defects in Tumorigenesis.

Chromosomal instability and its effect on cell lines.

Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.

Molecular landscape and subtype-specific therapeutic response of nasopharyngeal carcinoma revealed by integrative pharmacogenomics

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer type with high morbidity in Southeast Asia, however the pathogenic mechanism of this disease is poorly understood. Using integrative pharmacogenomics, we find that NPC subtypes maintain distinct molecular features, drug responsiveness, and graded radiation sensitivity. The epithelial carcinoma (EC) subtype is characterized by activations of microtubule polymerization and defective mitotic spindle checkpoint related genes, whereas sarcomatoid carcinoma (SC) and mixed sarcomatoid-epithelial carcinoma (MSEC) subtypes exhibit enriched epithelial-mesenchymal transition (EMT) and invasion promoting genes, which are well correlated with their morphological features. Furthermore, patient-derived organoid (PDO)-based drug test identifies potential subtype-specific treatment regimens, in that SC and MSEC subtypes are sensitive to microtubule inhibitors, whereas EC subtype is more responsive to EGFR inhibitors, which is synergistically enhanced by combining with radiotherapy. Through combinational chemoradiotherapy (CRT) screening, effective CRT regimens are also suggested for patients showing less sensitivity to radiation. Altogether, our study provides an example of applying integrative pharmacogenomics to establish a personalized precision oncology for NPC subtype-guided therapies.

Mitotic checkpoint defects: en route to cancer and drug resistance.

Loss of mitosis regulation is a common feature of malignant cells that leads to aberrant cell division with inaccurate chromosome segregation. The mitotic checkpoint is responsible for faithful transmission of genetic material to the progeny. Defects in this checkpoint, such as mutations and changes in gene expression, lead to abnormal chromosome content or aneuploidy that may facilitate cancer development. Furthermore, a defective checkpoint response is indicated in the development of drug resistance to microtubule poisons that are used in treatment of various blood and solid cancers for several decades. Mitotic slippage and senescence are important cell fates that occur even with an active mitotic checkpoint and are held responsible for the resistance. However, contradictory findings in both the scenarios of carcinogenesis and drug resistance have aroused questions on whether mitotic checkpoint defects are truly responsible for these dismal outcomes. Here, we discuss the possible contribution of the faulty checkpoint signaling in cancer development and drug resistance, followed by the latest research on this pathway for better outcomes in cancer treatment.

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.

PO-466 PI3K-C2A regulates mitotic spindle assembly and chemotherapy response in breast cancer

IntroductionProper organisation of the mitotic spindle is key to genetic stability but the molecular components of inter-microtubule (MT) bridges that crosslink kinetochore fibres (K-fibres) are still largely unknown. Here, we identify class II phosphoinositide 3-OH kinase a (PI3K-C2α) as a limiting scaffold protein organising the clathrin and TACC3 complex crosslinking K-fibres.Material and methodsPik3c2a+/- mice were intercrossed with a transgenic strain expressing the activated HER-2/Neu oncogene in the mammary gland. Mice were weekly followed for survival, tumour appearance and growth. Primary Murine Mammary Epithelial Tumour (MMET) cells were derived from early and late stage tumours. Truncating PI3KC2α mutants were generated and interaction with TACC3 was tested. Levels of PI3K-C2α expression were assessed by IHC in breast cancer tissue microarrays (TMA) and correlated with response to chemotherapy.Results and discussionsLoss of PI3K-C2α expression is a frequent occurrence in breast cancer patients (48%) and correlates with local recurrence and metastatic disease. The heterozygous loss of PI3K-C2α initially delays tumour onset but, on the long run, leads to the convergent evolution of aggressive clones with mitotic checkpoint defects. In line with this, downregulation of PI3K-C2α promotes spindle alterations and aneuploidy, indicating that PI3K-C2α expression is a key determinant of genomic stability. As a consequence of the altered spindle, reduction of PI3K-C2α expression increases the sensitivity to anti-MT drugs, such as paclitaxel, in pre-clinical models and in breast cancer patients.ConclusionLoss of PI3K-C2α expression is a frequent occurrence in breast cancer patients (48%) and correlates with local recurrence and metastatic disease. The heterozygous loss of PI3K-C2α initially delays tumour onset but, on the long run, leads to the convergent evolution of aggressive clones with mitotic checkpoint defects. In line with this, downregulation of PI3K-C2α promotes spindle alterations and aneuploidy, indicating that PI3K-C2α expression is a key determinant of genomic stability. As a consequence of the altered spindle, reduction of PI3K-C2α expression increases the sensitivity to anti-MT drugs, such as paclitaxel, in pre-clinical models and in breast cancer patients.

Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in many countries in south-eastern Asia. Chromosomal abnormalities have been commonly found in NPC, but the underlying mechanism is not well understood. We determined mitotic indices, the staining pattern of nuclear DNA and cell cycle profiles of NPC cells in response to treatment with microtubule-disrupting agents, and found that the mitotic checkpoint was defective in two out of five (40%) of the tested NPC cell lines. We also observed that an aberrantly reduced expression of MAD2, one of the key components of mitotic checkpoint, correlated with the loss of checkpoint control. Our findings suggest that a defective mitotic checkpoint characterized by reduced expression of MAD2 contributes to chromosomal instability in NPC.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Corrigendum: Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells.

Corrigendum: Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells.

Abstract A11: Cyclin A expression and endocrine resistance in an experimental model of murine mammary carcinomas

Abstract Most of human breast cancers are aneuploid because of chromosome missegregation occurring during mitosis. Deregulation in the control of cell cycle progression is frequently found in mammary carcinomas and is associated with mitotic spindle checkpoint defects and aneuploidy. The Laboratory of Hormonal Carcinogenesis has developed an experimental model of mammary carcinomas by the administration of medroxyprogesterone acetate (MPA) to BALB/c mice. These tumors are metastatic luminal ductal carcinomas, which express estrogen and progesterone receptors (PR) and show different responses to antiprogestin treatment. An association between PR isoform expression and endocrine therapy resistance was found in these mammary carcinomas. Tumors with higher levels of isoform A of PR (PRA) than isoform B (PRB) are inhibited by antiprogestin treatment while those with the opposite ratio are antiprogestin resistant tumors. The cytogenetic analysis of these MPA-induced mammary carcinomas showed that all the diploid mammary tumors respond to antiprogestin treatment, whereas aneuploid mammary tumors can be responsive or resistant to the endocrine therapy. Taking into account that Cyclin A has been related to chromosome instability and has also been involved in PR activation, in this study we evaluated its role in aneuploidy and hormone resistance using the MPA breast cancer model. We analyzed the Cyclin A expression by Western blot (WB) and immunohistochemistry (IHC) in the hormone dependent and the independent (HI) derived variants with different antiprogestin responsiveness and ploidy levels. WB studies revealed for Cyclin A two bands of estimated molecular weights of 52 kDa and 48 kDa in all the mammary tumors analyzed. The 52 kDa form of Cyclin A was overexpressed in the aneuploid unresponsive C4-2-HI tumor (10 fold; p<0.05), whereas the 48 kDa Cyclin A form was overexpressed in the resistant C4-HIR tumor (6 fold; p<0.05), compared to the responsive tumors in the C4 tumor family. On the other hand, both forms of Cyclin A were overexpressed in the resistant C7-2-HIR tumor (52 kDa: 6 fold; 48 kDa: 1.7, p<0.05), while the unresponsive C7-HI tumor expressed higher levels of the 48 kDa form (1.5 fold), as compared to the diploid responsive tumor. No differences were observed in the expression of the 48 kDa form of the cyclin in the mammary tumors of 59 tumor family, although the 52 kDa band was increased in one of the responsive HI tumors. These results were confirmed by IHC analysis of the paraffin sections of these tumors. A higher percentage of Cyclin A positive nuclei was observed in the resistant C4-HIR and C7-HI tumors, compared to the responsive tumors studied. These results suggest that in addition to its role in cell cycle control and its participation in aneuploidy, Cyclin A might be involved in the onset of antiprogestin resistance in the MPA breast cancer model. Citation Format: Melina Bilinski, Claudia Lanari, Victoria T. Fabris. Cyclin A expression and endocrine resistance in an experimental model of murine mammary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A11.

Mad1 at the Golgi apparatus: a story beyond kinetochores

Mad1 at the Golgi apparatus: a story beyond kinetochores

Induction of chromosome instability and stomach cancer by altering the expression pattern of mitotic checkpoint genes in mice exposed to areca-nut

BackgroundThere are strong indications for a causal association between areca-nut consumption and cancers. In Meghalaya, India, the variety of areca-nut is used as raw and unprocessed form whose chemical composition and pharmacological actions have been reported. Yet we know little on the initial pathway involved in areca-nut associated carcinogenesis since it is difficult to assess its effects on genetic alterations without interference of other compounding factors. Therefore, present study was undertaken in mice to verify the ability of raw areca-nut (RAN) to induce cancer and to monitor the expression of certain genes involved in carcinogenesis. This study was not intended to isolate any active ingredients from the RAN and to look its action.MethodsThree groups of mice (n = 25 in each) were taken and used at different time-points for different experimental analysis. The other three groups of mice (n = 15 in each) were considered for tumor induction studies. In each set, two groups were administered RAN-extract ad libitum in drinking water with or without lime. The expression of certain genes was assessed by conventional RT-PCR and immunoblotting. The mice were given the whole RAN-extract with and without lime in order to mimic the human consumption style of RAN.ResultsHistological preparation of stomach tissue revealed that RAN induced stomach cancer. A gradual increase in the frequency of precocious anaphase and aneuploid cells was observed in the bone marrow cells with a greater increment following RAN + lime administeration. Levels of p53, Bax, Securin and p65 in esophageal and stomach cells were elevated during early days of RAN exposure while those of different mitotic checkpoint proteins were downregulated. Apoptotic cell death was detected in non-cancerous stomach cells but not in tumor cells which showed overexpression of Bax and absence of PARP.ConclusionPresent study suggested (a) RAN induces stomach cancer, however, presence of lime promoted higher cell transformation and thereby developed cancer earlier, (b) perturbations in components of the chromosome segregation machinery could be involved in the initial process of carcinogenicity and (c) the importance of precocious anaphase as a screening marker for identification of mitotic checkpoint defects during early days.

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 < 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< 0.01) and aberrant chromosome numbers (P < 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

Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells

Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells

Bone morphogenetic protein (BMP) signaling regulates mitotic checkpoint protein levels in human breast cancer cells

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

The role of MOF in the ionizing radiation response is conserved in Drosophila melanogaster.

In Drosophila, males absent on the first (MOF) acetylates histone H4 at lysine 16 (H4K16ac). This acetylation mark is highly enriched on the male X chromosome and is required for dosage compensation in Drosophila but not utilized for such in mammals. Recently, we and others reported that mammalian MOF, through H4K16ac, has a critical role at multiple stages in the DNA damage response (DDR) and double-strand break repair pathways. The goal of this study was to test whether mof is similarly required for the response to ionizing radiation (IR) in Drosophila. We report that Drosophila mof mutations in males and females, as well as mof knockdown in SL-2 cells, reduce post-irradiation survival. MOF depletion in SL-2 cells also results in an elevated frequency of metaphases with chromosomal aberrations, suggesting that MOF is involved in DDR. Mutation in Drosophila mof also results in a defective mitotic checkpoint, enhanced apoptosis, and a defective p53 response post-irradiation. In addition, IR exposure enhanced H4K16ac levels in Drosophila as it also does in mammals. These results are the first to demonstrate a requirement for MOF in the whole animal IR response and suggest that the role of MOF in the response to IR is conserved between Drosophila and mammals.

A mad partner for Shugoshin in meiosis

The centromeric cohesin protector Shugoshin (Sgo) and the spindle checkpoint protein Mad2 are known to act in distinct pathways for ensuring accurate chromosome segregation during eukaryotic cell division. In this issue of The EMBO Journal , [Orth et al (2011)][1] describe an unexpected physical interaction between vertebrate Sgo and Mad2, as well as indications of their potential mutual regulation in meiosis. [1]: #ref-7

Abstract LB-267: Whole-genome and transcriptome interrogation of metastatic chemo-resistant triple negative breast cancer from African American patients

Abstract Introduction: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and Her2-neu, and accounts for ∼15% of all breast cancer diagnoses. Adjuvant chremotherapy and surgery can be effective in many women, although relapse with chemoresistant tumors that are difficult to treat is common. Importantly, these tumors may disproportionately affect African American women. Here, we present initial integrated analysis of matched normal and tumor whole genome and tumor transcriptome sequencing data from three African American patients with metastatic chemo-resistant TNBC, to better understand the compendium of somatic events occurring in these tumors within this high-risk population. Methods: Next Generation Sequencing was performed using the SOLiD version 4.0 system. Patient matched tumor and germline genomes are sequenced to 300 million mappable mate-pair reads (30X) and analyzed using custom paired analysis tools to uncover somatic alterations. Tumor transcriptomes are sequenced (RNA-seq) to 30 million uniquely aligned reads using Life Technologies Bioscope. For expression analysis, patient RNA-seq data are compared to data generated from ethnicity-matched population-based control hyperplastic breast tissue using EdgeR and DEGseq. Results: On average, we detected >3 million germline variants in these three African American patients. Review of germline results also provides information on BRCA1/2 status. Somatic paired analysis has revealed point mutations, small indels, copy number alterations, and translocations, in known cancer genes, and has revealed potential novel genes as well. Among three patients sequenced thus far, we have uncovered both unique and common genomic and transcriptomic perturbations. In both patients, unique somatic genomic alterations are associated with upregulation of specific signaling pathways. RNA-seq-based expression analysis has revealed a profile dominated by perturbations in cell cycle mitotic checkpoint defects. Furthermore, integrated analysis has provided key insights into the transcriptional consequences of a number of genomic alterations including exon skipping events and the discovery of potential fusion transcripts. Importantly, DNA and RNA changes have been validated by independent methods and technologies. Conclusions: Deep genome and transcriptome profiling of chemo-resistant TNBC has provided insights into events occurring in this difficult to treat cancer. These data are being leveraged to provide an opportunity for informed therapeutic options for intervention of this difficult to treat form of cancer. 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-267. doi:10.1158/1538-7445.AM2011-LB-267

Alterations of mitotic checkpoint genes play a role in embryonic chromosome mosaicism

OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts.DESIGN: Experimental study.MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (≥Grade 3BB) (n=21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR.RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n=8 euploid, Group B, n=8 uniform aneuploidy (meiotic error) and Group C, n=5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes.CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome. OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts. DESIGN: Experimental study. MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (≥Grade 3BB) (n=21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR. RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n=8 euploid, Group B, n=8 uniform aneuploidy (meiotic error) and Group C, n=5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes. CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome.