Spindle Assembly Checkpoint Genes Research Articles

TMOD-01. EGFR AMPLIFICATION ACCOMPANIES ADAPTATION TO MITOGEN-INDUCED DEFECTIVE MITOSIS IN PDGFA

Abstract Using a newly described in vitro murine model of GBM [Bohm et al., Neuro-Oncol 2020 and Omairi et al., Neuro-Oncol 2023] in which P53 null neural progenitor cells (NPCs) divide abnormally and evolve a GBM-like genome during exposure to Platelet-Derived Growth Factor-AA (PDGFA), we asked how a brain-abundant mitogen could transform NPCs. By analyzing gene and protein expression over time, we found that PDGFA fails to induce the transcription of kinetochore and spindle assembly checkpoint genes, while simultaneously driving NPCs to enter mitosis. These dual effects caused chromosome miss-segregation in continuously dividing NPCs; moreover, they occurred in both WT and null NPCs, although only null cells survived defective mitosis. These surviving cells gradually expanded in PDGFA accumulating both random and clonal chromosomal re-arrangements. Transcriptome analysis of NPCs in PDGFA revealed significant under-expression of Foxm1, the major regulator of kinetochore transcription, together with over-expression and phosphorylation of the immediate early response gene, FOS. Analysis of signalling downstream of PDGFRα identified the Ras-MAPK pathway, especially ERK, as responsible for FOS activation. As surviving null cells gradually expanded, they accumulated random and recurrent chromosomal rearrangements. Expansion and subsequent PDGFA-independent proliferation and tumorigenicity were associated with re-expression of Foxm1 and kinetochore proteins, and accompanied by over-expression of Egfr, an RTK-signature that defines human GBM. By stimulating proliferation without setting the stage for error-free mitosis, exposure to PDGFA transforms p53 null NPCs and generates Egfr amplified GBM-like cancer cells.

Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.

Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Evidence of a SAC response by several of these proteins is found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes does not induce a SAC response. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is silenced. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal, or "streaming", is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that dynein adaptor Spindly is also required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

A whole-genome CRISPR screen identifies the spindle accessory checkpoint as a locus of nab-paclitaxel resistance in a pancreatic cancer cell line

Pancreatic adenocarcinoma is one of the most aggressive and lethal forms of cancer. Chemotherapy is the primary treatment for pancreatic cancer, but resistance to the drugs used remains a major challenge. A genome-wide CRISPR interference and knockout screen in the PANC-1 cell line with the drug nab-paclitaxel has identified a group of spindle assembly checkpoint (SAC) genes that enhance survival in nab-paclitaxel. Knockdown of these SAC genes (BUB1B, BUB3, and TTK) attenuates paclitaxel-induced cell death. Cells treated with the small molecule inhibitors BAY 1217389 or MPI 0479605, targeting the threonine tyrosine kinase (TTK), also enhance survival in paclitaxel. Overexpression of these SAC genes does not affect sensitivity to paclitaxel. These discoveries have helped to elucidate the mechanisms behind paclitaxel cytotoxicity. The outcomes of this investigation may pave the way for a deeper comprehension of the diverse responses of pancreatic cancer to therapies including paclitaxel. Additionally, they could facilitate the formulation of novel treatment approaches for pancreatic cancer.

Heat stress during male meiosis impairs cytoskeletal organization, spindle assembly and tapetum degeneration in wheat.

The significance of heat stress in agriculture is ever-increasing with the progress of global climate changes. Due to a negative effect on the yield of staple crops, including wheat, the impairment of plant reproductive development triggered by high ambient temperature became a restraint in food production. Although the heat sensitivity of male meiosis and the following gamete development in wheat has long been recognized, a detailed structural characterization combined with a comprehensive gene expression analysis has not been done about this phenomenon. We demonstrate here that heat stress severely alters the cytoskeletal configuration, triggers the failure of meiotic division in wheat. Moreover, it changes the expression of genes related to gamete development in male meiocytes and the tapetum layer in a genotype-dependent manner. 'Ellvis', a heat-tolerant winter wheat cultivar, showed high spikelet fertility rate and only scarce structural aberrations upon exposure to high temperature. In addition, heat shock genes and genes involved in scavenging reactive oxygen species were significantly upregulated in 'Ellvis', and the expression of meiosis-specific and major developmental genes showed high stability in this cultivar. In the heat-sensitive 'Mv 17-09', however, genes participating in cytoskeletal fiber nucleation, the spindle assembly checkpoint genes, and tapetum-specific developmental regulators were downregulated. These alterations may be related to the decreased cytoskeleton content, frequent micronuclei formation, and the erroneous persistence of the tapetum layer observed in the sensitive genotype. Our results suggest that understanding the heat-sensitive regulation of these gene functions would be an essential contribution to the development of new, heat-tolerant cultivars.

Deficiency in homologous recombination is associated with changes in cell cycling and morphology in Saccharomyces cerevisiae

Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated internally by chemicals and enzymes, in the absence of exogenous agents, though the sources and consequences of such endogenously generated DSBs remain poorly understood. In the current study, we have investigated the impact of reduced recombinational repair of endogenous DSBs on stress responses, cell morphology and other physical properties of S. cerevisiae (budding yeast) cells. Use of phase contrast and DAPI-based fluorescence microscopy combined with FACS analysis confirmed that recombination-deficient rad52 cell cultures exhibit chronically high levels of G2 phase cells. Cell cycle phase transit times during G1, S and M were similar in WT and rad52 cells, but the length of G2 phase was increased by three-fold in the mutants. rad52 cells were larger than WT in all phases of the cycle and displayed other quantifiable changes in physical characteristics. The high G2 cell phenotype was abolished when DNA damage checkpoint genes, but not spindle assembly checkpoint genes, were co-inactivated with RAD52. Several other RAD52 group mutants (rad51, rad54, rad55, rad57 and rad59) also exhibited the high G2 cell phenotype. The results indicate that recombination deficiency leads to accumulation of unrepaired DSBs during normal mitotic growth that activate a major stress response and produce distinct changes in cellular physiology and morphology.

Budding uninhibited by benzimidazoles 1 might be a poor prognosis biomarker promoting the progression of papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is one of the most widespread malignant tumors of the endocrine system, with a high incidence. Budding uninhibited by benzimidazoles 1 (BUB1), one of the spindle assembly checkpoint (SAC) genes, is a multitask protein kinase required for eukaryotic chromosome segregation. Although BUB1 has been explored in several types of cancer, its biological role and molecular mechanisms in PTC remain unclear. In this study, we performed an examination of four public datasets along with local PTC cohorts and discovered that BUB1 was elevated in PTC compared to non-cancer tissues. High BUB1 expression was linked with the status of BRAFV600E , RAS, and TERT after statistical analysis. Clinically, BUB1 is associated with a variety of clinicopathological features in PTC patients. Interestingly, analysis of the TCGA database showed that BUB1 was closely associated with poor prognosis of PTC and significantly correlated with PFS. As determined by regression analysis, BUB1, and T stage were independent predictors of PTC and were related to BRAFV600E and lymph node metastatic status. By RT-qPCR, BUB1 was considerably overexpressed in PTC cell lines in comparison with normal thyroid epithelial cells. We confirmed that the knockdown of BUB1 in BCPAP and TPC1 cell lines significantly inhibited cell proliferation, cloning, and migration in vitro experiments. These results imply that BUB1 may be a significant oncogenic gene that is directly associated with the prognosis of PTC and may represent a future target for therapeutic intervention.

Mitogen-induced defective mitosis transforms neural progenitor cells.

Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.

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.

Upregulated mitosis-associated genes CENPE, CENPF, and DLGAP5 predict poor prognosis and chemotherapy resistance of Acute Myeloid Leukemia.

Mitosis-associated genes are dysregulated in many types of cancers and play important roles in disease progression and chemotherapy resistance. However, their expression and functions in chemotherapy-resistant Acute Myeloid Leukemia (AML) are still largely undetermined. This study aims to explore the roles of spindle assembly checkpoint (SAC) genes CENPE, CENPF, and DLGAP5 in chemotherapy-resistant AML. RNA-sequencing (RNA-seq) was performed in patients with chemotherapy-resistant AML and chemotherapy-sensitive AML. AML mRNA data from 151 patients with recurrence were downloaded from TCGA. Integrated analysis of the differentially expressed genes (DEGs), GO and KEGG pathways. CENPE, CENPF, or DLGAP5 knockdown cell lines were used to analyse proliferation, apoptosis and cell cycle alterations. A total of 87 DEGs (48 upregulated and 39 downregulated) were obtained through gene analysis of R/R-AML and a total of 329 DEGs (202 upregulated and 127 downregulated) were obtained in refractory S-AML. Upregulated DEGs were mainly enriched in cell cycle (GO: 0007049, hsa04110) and mitotic cell cycle (GO: 0000278) processes and pathway. Venn diagram analysis identified the most upregulated DEGs (including CENPE, CENPF, and DLGAP5) in chemoresistant AML. The expression of CENPE, CENPF and DLGAP5 in R-AML (TCGA) was significantly higher than that of primary AML (GEO). The proliferation of K562 cells after CENPE and DLGAP5 knockdown was significantly decreased (P= 0.0001 and P= 0.0006). In THP-1 cells, the CCK-8 values after CENPE, CENPF and DLGAP5 knockdown were significantly decreased (P= 0.01, P= 0.0395 and P= 0.0362). Knockdown of CENPE, CENPF and DLGAP5 significantly increased cell apoptosis by regulating Caspase-9, BAX, TP-53 and bcl-2, and induced cell cycle arrested by regulating CDK1, CDK2, CDKN1A, and CyclinD1. In conclusion, the mitotic cell cycle-associated genes CENPE, CENPF, and DLGAP5 were upregulated in chemotherapy-resistant AML patients and might be useful for predicting poor prognosis.

NudCD1 as a prognostic marker in colorectal cancer and its role in the upregulation of cellular spindle assembly checkpoint genes and LIS1 pathways

ObjectiveTo investigate the role of NudCD1 in spindle assembly checkpoint regulation and in the prognosis of colorectal cancer. MethodsImmunohistochemical staining was used to detect in situ expression of NudCD1 in 100 colorectal cancer tissue samples. A chi-square test was used to analyse the correlation between the NudCD1 protein expression level of the cancer tissues and clinicopathological features. The Kaplan–Meier survival analysis was used to assess the correlation between the NudCD1 mRNA expression and the three-year survival of patients with colorectal cancer. The impact of NudCD1 on the development of colorectal cancer and the underlying molecular mechanisms were assessed by flow cytometry cell cycle and apoptosis assays after lentiviral overexpression of NudCD1 in two colorectal cancer cell lines. Quantitative real-time PCR was used to assess mRNA expression of the cellular spindle assembly checkpoint genes BUB1, BUBR1, MAD1, CDC20 and MPS1, as well as the downstream genes LIS1, DYNC1H1, and DYNLL1 in the NudC/LIS1/dynein pathway.ResultsCompared with normal intestinal tissue (8.00% with high expression), the expression of NudCD1 protein in colorectal cancer tissue was significantly higher (58.00% with high expression, P < 0.01). In addition, expression of NudCD1 significantly correlated with the degree of tumour differentiation and the TNM staging (P < 0.01), as well as the depth of invasion of the primary tumour and lymph node metastasis (P < 0.05). However, there was no correlation with gender, age, tumour site, gross type, tumour size or distant metastasis. The Kaplan–Meier survival analysis showed that patients with high NudCD1 expression in colorectal cancer tissues had a significantly shorter survival time than those with low expression of NudCD1 (P < 0.01). Compared with the transfection of the empty vector, colon cancer HT-29 cells with overexpressed NudCD1 had significantly increased mRNA levels of BUBR1, MPS1 and LIS1. The DNA synthesis phase (S phase) was significantly shorter in cells overexpressing NudCD1 than in the control group (43.83% ± 1.57%, P < 0.05), while there was no difference in apoptosis in the two groups.ConclusionNudCD1 can serve as a valuable prognostic marker for colorectal cancer. It may be involved in the regulation of spindle-assembly checkpoint-gene expression and the LIS1 pathway of colorectal cancer cells.

Abstract 6057: Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb

Abstract We have previously reported (Int J Radiat Oncol Biol Phys. 107(1):194-201, 2020) that human GBM-stem like cells (GSCs) implanted into the right striatum of nude mice migrate/invade throughout the right hemisphere (RH) as well as into the olfactory bulb (OB). The highly invasive nature of the GSCs suggested a model system for evaluating the effects of specific brain microenvironments on glioma radioresponse and the potential for topographic heterogeneity in radioresistance. Subsequent analyses revealed that GSCs in the OB were significantly more radioresistant than those in the RH. Because the OB is highly enriched in neurons and given recent data suggesting that neuron/glioma interactions drive GBM growth, in this study we first defined contact between GSCs and neurons in the OB and RH using immunohistochemistry (IHC) and confocal microscopy. In the OB 56% of tumor cells were in direct contact with neurons as compared to 24% of tumor cells in the RH. Moreover, among the tumor cells in contact with neurons 25% had 2 or more neuronal contacts in the OB versus 2% in the RH. These data suggest a significantly greater level of glioma-neurons interaction in the OB. To further investigate the OB as a radioresistant niche, RNAseq was used to define the transcriptome of NSC11 GSCs located in the OB versus the RH. Of the greater than 15,000 genes evaluated, 1887 genes were differentially expressed between the 2 sites: 872 upregulated and 1015 down regulated in the OB versus the RH. Functional analyses (IPA and GSEA) indicated that among the genes overexpressed in the OB GSCs were a group associated with the cell cycle, specifically mitosis and the spindle assembly checkpoint (SAC). Comparison of the SAC genes overexpressed in the OB with GBM clinical outcomes (TCGA) showed a significant correlation with decreased patient survival. These analyses suggested that cell cycle regulation may play a role in the radioresistance of OB GSCs. Accordingly, radiation-induced cell cycle redistribution of GSCs in the OB and the RH was determined by IHC analysis of cell cycle phase markers and flow cytometry (propidium iodide) performed on dissociated tumor cells. Results showed a significantly higher percentage of cells in G2 in the OB at 24h and 48h after 10Gy, suggestive of prolonged G2 arrest. In addition, analysis of radiation-induced DNA DSBs using γH2AX and 53BP1 nuclear foci showed faster and more complete foci dispersal after 10Gy in the OB GSCs versus those in the RH, indicative of increased DSBs repair in the OB. These data suggest that GSC radioresistance in the OB is the result of a more effective DNA damage response. Moreover, results suggest that the murine OB may provide a model system for investigating GBM radioresistance as well as the potential role of glioma/neuron interactions. Citation Format: Charlotte M. Degorre, Ian C. Sutton, Stacey L. Lehman, Uma T. Shankavaram, Kevin A. Camphausen, Philip J. Tofilon. Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6057.

Abstract 2269: The spindle assembly checkpoint as a therapeutic vulnerability in RIT1-mutant lung adenocarcinoma

Abstract Standard care of lung cancer is moving away from chemotherapy in favor of personalized approaches based on specific mutations in each tumor. Genome-sequencing studies have identified somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) in lung adenocarcinoma patients. Of the identified mutations, M90I is the most recurrent. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven disease could greatly improve patient outcomes. Little is known about how RIT1 drives cellular transformation. To genetically dissect signaling pathways downstream of RIT1, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells in which cell survival is dependent on expression of RIT1M90I. We found that RIT1-mutant cells were highly dependent on components of the Spindle Assembly Checkpoint (SAC), including the Aurora kinases (A and B). The SAC is a surveillance mechanism that ensures proper chromosome segregation during mitosis. We hypothesized that RIT1M90I weakened the SAC and rendered cells vulnerable to loss of mitotic regulators. To explore this, we performed time-lapse imaging in HeLa H2B-GFP cells stably expressing RIT1M90I. In parental cells, the median duration of mitosis was 70.5 min (95% CI = 63 – 82 min), while in RIT1M90I-mutant cells, this was reduced to 48 min (95% CI = 45 – 51 min). Mitotic index was unaffected, suggesting that RIT1M90I does not regulate mitotic entry. This difference in mitotic timing was eliminated by treatment with reversine, an inhibitor that abolishes the SAC, demonstrating that RIT1M90I perturbs mitotic timing at the level of the SAC. If RIT1M90I weakens the SAC, we would expect higher prevalence of chromosomal abnormalities (such as chromosome bridges) in RIT1M90I-mutant cells. Indeed, analysis of fixed-cell populations indicated that RIT1M90I-mutant cells showed higher prevalence of mitotic abnormalities (79% of RIT1M90I-mutant cells compared to 46% of parental cells, 95% CI= 15 – 53). These data support the hypothesis that RIT1M90I weakens the SAC and imply that further SAC perturbation could be lethal. To explore this, we performed a small molecule screen of 160 clinically-relevant inhibitors in PC9-RIT1M90I and PC9-KRASG12V cells. Intriguingly, RIT1M90I-mutant cells were more sensitive than RAS-mutant cells to alisertib and barasertib, inhibitors of Aurora kinase A and B, respectively. Furthermore, alisertib or barasertib treatment abrogated soft agar colony formation in RIT1M90I-mutant cells but not in RAS-mutant cells. Together with our functional genomic analysis, we propose a model whereby expression of RIT1M90I weakens the SAC, thereby accelerating mitosis, increasing the abundance of mitotic abnormalities, and rendering cells vulnerable to genetic knockdown of SAC genes or small molecule inhibition of Aurora kinases A/B. Citation Format: Amanda Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. The spindle assembly checkpoint as a therapeutic vulnerability in RIT1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2269.

Spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival.

The study aimed to investigate the role of spindle assembly checkpoint (SAC) in cancer cells with compromised genomic integrity. Chromosomal instability (CIN) gives cancer cells an adaptive advantage. However, maintaining the balance of this instability is crucial for the survival of cancer cells as it could lead them to the mitotic catastrophe. Therefore, cancer cells adapt to the detrimental effects of CIN. We hypothesized that changes in SAC might be one such adaptation mechanism. The focus of the study was BUB1B, an integral part of the checkpoint. Clinical datasets were analyzed to compare expression levels of SAC genes in normal tissue vs. breast carcinoma. The effects of the reduction of BUB1B expression was examined utilizing RNA interference method with siRNAs. In vitro viability, clonogenicity, apoptosis, and SAC activity levels of a variety of breast cancer (BrCa) cell lines, as well as in vivo tumorigenicity of the triple-negative breast cancer (TNBC) cell line MDA-MB-468, were tested. Additionally, the chromosomal stability of these cells was tested by immunofluorescence staining and flow cytometry. In clinical breast cancer datasets, SAC genes were elevated in BrCa with BUB1B having the highest fold change. BUB1B overexpression was associated with a decreased probability of overall survival. The knockdown of BUB1B resulted in reduced viability and clonogenicity in BrCa cell lines and a significant increase in apoptosis and cell death. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown also impaired chromosome alignment and resulted in acute chromosomal abnormalities. We also showed that BUB1B knockdown on the MDA-MB-468 cell line decreases tumor growth in mice. A functional spindle assembly checkpoint is essential for the survival of BrCa cells. BUB1B is a critical factor in SAC, and therefore breast cancer cell survival. Impairment of BUB1B has damaging effects on cancer cell viability and tumorigenicity, especially on the more aggressive variants of BrCa.

Abstract 4387: hnRNP Q1 promotes cell proliferation and tumorigenesis by translationally up-regulating cell cycle-related genes through the EGF pathway

Abstract Heterogeneous nuclear ribonucleoprotein (hnRNP) Q1, an RNA-binding protein, has been reported as overexpressing in cancer cells. However, the role of hnRNP Q1 in tumorigenesis remains unclear. By using RNA-immunoprecipitation (IP) assay following next-generation sequencing, a group of cell cycle-related genes targeted by hnRNP Q1 were identified, including Aurora-A kinase. Overexpressed hnNRP Q1 can up-regulate Aurora-A and many spindle assembly checkpoint (SAC) proteins, but not alter their mRNA levels, through enhancing translational efficiency by interacting with the mRNA 5’-UTR. Ribosomal S6-IP and ribosomal profiling assay further confirmed the translational regulation of these mRNAs by hnRNP Q1. Stimulation with epidermal growth factor (EGF) enhances both binding between hnRNP Q1 and these mRNAs as well as the efficacy of the hnRNP Q1-induced translation of these mRNAs. EGF/hnRNP Q1-induced translation is mediated by the mTOR and ERK pathways. In addition, hnRNP Q1 overexpression is positively correlated with the levels of Aurora-A and these SAC genes in human colorectal cancer tissues. Taken together, our data suggest that hnRNP Q1 plays an important role in translationally regulating the expression of Aurora-A and a group of cell cycle-related genes, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer. It may thereby contribute to tumorigenesis by translationally up-regulating these genes in colorectal cancer. Citation Format: Liang-Yi Hung. hnRNP Q1 promotes cell proliferation and tumorigenesis by translationally up-regulating cell cycle-related genes through the EGF pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4387.

Aneuploidy: Cancer strength or vulnerability?

Aneuploidy is a very rare and tissue‐specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age‐related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell‐like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy‐tolerating mechanisms as cancer vulnerability.

The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes

Heterogeneous nuclear ribonucleoprotein (hnRNP) Q1, an RNA-binding protein, has been implicated in many post-transcriptional processes, including RNA metabolism and mRNA splicing and translation. However, the role of hnRNP Q1 in tumorigenesis remains unclear. We previously performed RNA immunoprecipitation (RIP)-seq analysis to identify hnRNP Q1-interacting mRNAs and found that hnRNP Q1 targets a group of genes that are involved in mitotic regulation, including Aurora-A. Here, we demonstrate that altering the hnRNP Q1 level influences the expression of the Aurora-A protein, but not its mRNA. Stimulation with epidermal growth factor (EGF) enhances both binding between hnRNP Q1 and Aurora-A mRNA as well as the efficacy of the hnRNP Q1-induced translation of Aurora-A mRNA. The EGF/hnRNP Q1-induced translation of Aurora-A mRNA is mediated by the mTOR and ERK pathways. In addition, we show that hnRNP Q1 up-regulates the translation of a group of spindle assembly checkpoint (SAC) genes. hnRNP Q1 overexpression is positively correlated with the levels of Aurora-A and the SAC genes in human colorectal cancer tissues. In summary, our data suggest that hnRNP Q1 plays an important role in regulating the expression of a group of cell cycle-related genes. Therefore, it may contribute to tumorigenesis by up-regulating the translation of these genes in colorectal cancer.

Folate deficiency induces mitotic aberrations and chromosomal instability by compromising the spindle assembly checkpoint in cultured human colon cells

Folates comprise the essential B9 vitamin that act as cofactors and cosubstrates in one-carbon metabolism for both biosynthesis and methylation of DNA and RNA. Folate deficiency (FD) has been shown to induce chromosomal instability (CIN), yet the underlying mechanisms are poorly understood. Here, we used human NCM460 colon mucosal cells as a model to investigate the effect of FD on spindle assembly checkpoint (SAC), a cell-cycle regulatory pathway preventing CIN during mitosis. Cells were maintained in medium containing 1.36 (FD) and 2260 nM (control, FC) folate for 21 days. CIN was measured by cytokinesis-block micronucleus assay; mitotic infidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of core SAC genes was examined by real-time quantitative PCR (RT-qPCR). We found that, relative to FC, FD significantly induced CIN in a time-dependent way (P < 0.01). Mitotic cells cultured in FD medium had significant higher frequencies of misalignment, misegregation and spindle multipolarity than those cultured in FC medium (P < 0.01). FD-induced SAC impairment and overriding, resulting premature mitotic exit and cell multinucleation (P < 0.05). Moreover, FD deregulated the expression of several key SAC genes (P < 0.01). Overall, these data are the first to demonstrate that FD substantially compromises SAC network which predisposes cells to mitotic aberrations and CIN. These results establish a new link between folate metabolism and SAC signalling, two pathways that are highly relevant for tumorigenesis.

Biphasic regulation of spindle assembly checkpoint by low and high concentrations of resveratrol leads to the opposite effect on chromosomal instability

Resveratrol (RSV) is a naturally occurring polyphenolic phytoalexin possessing numerous health-promoting effects. Chromosomal instability (CIN), usually results from defective spindle assembly checkpoint (SAC), is a major contributor to many diseases. While it’s recently recognized that RSV exhibits a nonlinear dose response for disease prevention, whether it’s the case for its role in CIN remains unknown. Here, we investigated the potential of a broad range of RSV concentrations (0.01–100μM) on CIN and the underlying mechanisms in human normal colon epithelial NCM460 cells. CIN was measured by cytokinesis-block micronucleus assay; mitotic fidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of SAC genes was examined by RT-qPCR. We found that 0.1μM RSV significantly reduced CIN (P<0.01), while 100μM RSV significantly induced it (P<0.05). Mitotic infidelity was significantly prevented by 0.1μM RSV but promoted by 100μM RSV (P<0.05 for both). Moreover, the function of SAC was sustained and impaired by 0.1μM and 100μM RSV, respectively. Several SAC genes, including Aurora-B, Aurora-C, Plk-1 and CENP-E, were significantly up-regulated and down-regulated by 0.1μM and 100μM RSV, respectively (P<0.05). In conclusion, RSV exhibited a biphasic dose-dependent effect on CIN that was exerted via the regulation of mitotic fidelity through the SAC network. The health implications of these findings were summarized.

Abstract 512: Knockdown of the spindle assembly checkpoint gene Bub1b results in increased cell death and cell cycle impairment in breast cancer

Abstract Genomic instability is an enabling hallmark of cancer that provides cancer cells a replicative advantage. Accumulation of genomic aberrations can also compromise the genomic integrity and put cells under mitotic stress. Maintaining balance between the instability that gives cancer cells a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial for survival of cancer cells. Mitotic catastrophe can be caused by rapid progression through mitosis before crucial checkpoints are met, resulting in cell death. To overcome this challenge, cancer cells may acquire overexpression of spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if they undergo mitosis prematurely. Bub1b is an important part of SAC and inhibits the onset of anaphase until all chromosomes are aligned at the metaphase plate. Our analysis of clinical datasets show Bub1b expression is elevated in breast cancer, compared to normal breast, and is exemplified by a pattern of increasing overexpression in more aggressive variants, such as triple negative breast cancer (TNBC). Bub1b overexpression also correlates with decreased overall survival in patients. Expression analysis of breast cancer cell lines corroborates this clinical data. We hypothesize that the requirement for Bub1b expression indicates a vulnerability of rapidly proliferating breast cancers cells, and the inhibition of Bub1b will result in cell death through mitotic catastrophe. Using RNA interference with siRNAs we knocked down Bub1b expression in a variety of breast cancer cells. This resulted in significant decrease in cellular viability and clonogenicity in soft agar. Furthermore, analyses of apoptosis using Annexin V and PI costaining showed that Bub1b knockdown results in significantly increased apoptosis and cell death, especially in the TNBC cell line MDA-MB-468. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. Bub1b knockdown resulted in silencing of SAC activity, as measured by Cyclin B levels, and also significantly impaired cell cycle progression in breast cancer cell lines. Additionally, knockdown of Bub1b in breast cancer cell lines demonstrated an additive effect when combined with DNA-damaging agents. Our data support that Bub1b is a critical player in breast cancer cell survival, by enabling them to overcome mitotic stress. We demonstrate that impairment of Bub1b has detrimental effects on cell cycle and progression of mitosis. Further investigation of the role of Bub1b in promoting successful chromosome alignment and proliferation of breast cancer cells with genomic instability could provide a novel pathway on how cancer overcomes the mitotic stress caused by genomic instability. These studies may also lead to novel therapeutic strategies that combine SAC inhibitors with standard genotoxic treatments. Citation Format: Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. Knockdown of the spindle assembly checkpoint gene Bub1b results in increased cell death and cell cycle impairment in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2017-512