Mitotic Chromosome Instability Research Articles

Abstract 5269: Evaluation of mitotic spindle inhibitors as cell autonomous inflammatory stimulants in MYC overexpressing breast cancers

Abstract The oncogenic transcription factor MYC is overexpressed in most cancer types, including difficult to treat human malignancies such as receptor triple-negative breast cancers (TNBC). MYC overexpression induces aneuploidy and suppresses anticancer immunity. Elevated levels of MYC can induce mitotic spindle assembly defects and chromosomal instability (CIN) events, exemplified by an increase in cells with micronuclei. These defects are partially mitigated by concomitant upregulation of various mitotic spindle genes. Micronuclei result in cytosolic DNA which can activate cGAS-STING signaling, activating an interferon regulatory transcriptional program responsible for mounting an inflammatory immune response that potentially enhances immune cell recognition. We hypothesized that pharmacological inhibition of specific MYC deregulated mitotic spindle genes will further exacerbate mitotic defects in MYC high cells resulting in synthetic-lethal cell death and additional CIN to activate robust cGAS-STING activation. Interferon activation can increase surface expression of antigen presentation machinery, such as MHC-I, restoring the immunogenicity of MYC high cells. Here, we used mouse breast tumor cells in which MYC expression is conditionally expressed as well as human breast cancer cell lines with different levels of MYC expression. We assessed the anti-proliferative and cytotoxic effects of 9 mitotic small molecular inhibitors that affect spindle function. cGAS-STING activation in inhibitor treated and untreated samples was measured by immunoblotting for pathway proteins phosphorylated TBK1, total TBK1, and IFIT1. Differences in MHC-I cell surface expression was quantified using flow cytometry. Our findings revealed that inhibition of kinesin-like protein KIFC1 and transforming acidic coiled-coil-containing protein 3 (TACC3) induced greater levels of apoptosis in MYC high models. Moreover, inhibition of KIFC1 robustly triggered cGAS-STING activity in MYC high cells as evidenced by increased protein abundance of phospho-TBK1 and IFIT1 in a dose-dependent manner. In addition, sublethal KIFC1 inhibition resulted in increased levels of MHC-I surface expression in MYC high models. Our findings suggest that inhibition of MYC regulated mitotic spindle genes, specifically KIFC1, can serve to preferentially eliminate MYC overexpressing aggressive cancers. Furthermore, sublethal concentrations of a KIFC1 inhibitor can stimulate a tumor cell-intrinsic inflammatory-pathway. Future studies will determine if targeting KIFC1 can enhance the response of MYC high cancers to immunotherapy. Citation Format: Mauricio Jacobo, Andrei Goga. Evaluation of mitotic spindle inhibitors as cell autonomous inflammatory stimulants in MYC overexpressing breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5269.

Changes in spindle morphology driven by TPX2 overexpression in MYC-driven breast cancer cells.

The MYC oncogene was previously shown to induce mitotic spindle defects, chromosome instability, and reliance on the microtubule-associated protein TPX2 to survive, but how TPX2 levels affect spindle morphology in cancer cells has not previously been examined in detail. We show that breast cancer cell lines expressing high levels of MYC and TPX2 possess shorter spindles with increased TPX2 localization at spindle poles. A similar effect was observed in non-transformed human RPE-1 cells compared to a tumor cell line (HeLa) that overexpresses MYC . These results demonstrate that TPX2 alters spindle length and morphology in cancer cells, which may contribute their ability to divide despite MYC-induced mitotic stress.

Disruption of CDK7 signaling leads to catastrophic chromosomal instability coupled with a loss of condensin-mediated chromatin compaction

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.

Atypical meningiomas with an immunohistochemical profile consistent with hypermetabolic or proliferative molecular groups show high mitotic index, chromosomal instability, and higher recurrence risk

The use of adjuvant radiotherapy is controversial for atypical meningiomas undergoing gross total resection. It has recently been proposed that meningiomas may be classified into four molecular groups (MG): immunogenic (MG1), benign NF2-wildtype (MG2), hypermetabolic (MG3), and proliferative (MG4). The two latter have the worst prognosis, and it has been suggested that they can be identified using ACADL and MCM2 immunostainings. We studied 55 primary atypical meningiomas, treated with gross total resection and no adjuvant therapies, to assess whether ACADL and MCM2 immuno-expression may identify patients at higher recurrence risk, thus requiring adjuvant treatments. Twelve cases resulted ACADL-/MCM2-, 9 ACADL + /MCM2-, 17 ACADL + /MCM2 + , and 17 ACADL-/MCM2 + . MCM2 + meningiomas displayed more frequent atypical features (prominent nucleoli, small cells with high nuclear-to-cytoplasmic ratio) and CDKN2A hemizygous deletion (HeDe) (P = 0.011). The immunoexpression of ACADL and/or MCM2 was significantly associated with higher mitotic index, 1p and 18q deletions, increased recurrence rate (P = 0.0006), and shorter recurrence-free survival (RFS) (P = 0.032). At multivariate analysis, carried out including ACADL/MCM2 immuno-expression, mitotic index, and CDKN2A HeDe as covariates, this latter resulted a significant and independent prognosticator of shorter RFS (P = 0.0003).

RNA-binding protein RNPC1 acts as an oncogene in gastric cancer by stabilizing aurora kinase B mRNA

BackgroundRNPC1 is reported to act as a tumor suppressor by binding and regulating the expression of target genes in various cancers. However, the role of RNPC1 in gastric cancer and the underlying mechanisms are still unclear. MethodsGastric cancer cells were stably transfected with lentivirus. Proliferation, migration, invasion, cell cycle in vitro and tumorigenesis in vivo were used to assess the role of RNPC1. Quantitative real-time PCR, western blotting and immunohistochemistry were used to detect the relationship between RNPC1 and aurora kinase B (AURKB). RNA immunoprecipitation (RIP), RNA electrophoretic mobility shift assays (REMSAs), and dual-luciferase reporter assays were used to identify the direct binding sites of RNPC1 with AURKB mRNA. A CCK-8 assay was conducted to confirm the function of AURKB in RNPC1-induced growth promotion. ResultsHigh RNPC1 expression was found in gastric cancer tissues and cell lines and was associated with high TNM stage. RNPC1 overexpression significantly promoted the proliferation, migration, and invasion of gastric cancer cells. Knockdown of RNPC1 could impede gastric cancer tumorigenesis in nude mice. AURKB expression was positively related to RNPC1. RNPC1 directly binds to the 3'-untranslated region (3'-UTR) of AURKB and enhances AURKB mRNA stability. AURKB reversed the proliferation induced by RNPC1 in gastric cancer cells. RNPC1 resulted in mitotic defects, aneuploidy and chromosomal instability in gastric cancer cells, similar to AURKB. ConclusionRNPC1 acts as an oncogene in gastric cancer by influencing cell mitosis by increasing AURKB mRNA stability, which may provide a potential biomarker and a therapeutic target for gastric cancer.

Study of mangal based naphthoquinone derivatives anticancer potential towards chemo-resistance related Never in mitosis gene A-related kinase 2-Insilico approach

ABSTRACT Never in mitosis gene A-related kinase 2 (NEK2) is a member of serine-threonine kinase mainly involved in cell cycle process. Clinical studies revealed that NEK2 overexpression in many tumour types, also reported for their association with genetic abnormalities like mitotic machinery deregulation and chromosomal instability. Besides NEK2 plays a key role in maintaining the transmuted phenotype of tumour cells and induction of chemo-resistance in tumour types. Our studies involved NEK2 transcriptional profile search, screening of druggable cavities in NEK2, Drug likeliness of mangrove derived naphthoquinone derivatives avicennone, avicennone A, avicennone B, avicennone C, avicennone D, avicenone E, avicennone F and avicennone G, avicequinone A, stenocarpoquinone B, avicequinone C, avicenol A, avicenol C, brugine, apigenin, chrysin and molecular docking studies to assess compounds binding efficacy towards NEK2. Irreversible inhibitors have several advantages over noncovalent inhibitors, comprising sustained pharmacodynamics with high potency practical application, and specificity. Thus Targeting the cysteine 22 (Cys-22) residue near the catalytic site of NEK2 kinase will result in irreversible inhibition. Avicenol A, Avicennone G, Chrysin and Brugine formed the irreversible covalent binding with NEK2 through Cys 22, thus they can be considered as potential kinase inhibitors with limited off-target response. But proposed compounds need to be tested further through in vitro and in vivo studies to propose as potent NEK2 inhibitors.

Identifying Novel Genes Involved in Dysregulation of Homologous Recombination and Genomic Integrity in Myeloma

Genomic instability leads to ongoing acquisition of genomic changes which underlie development and progression of cancer. We have previously demonstrated that homologous recombination (HR), the most precise DNA repair mechanism, is dysregulated in myeloma and contributes to genomic instability and development of drug resistance. Purpose of this study was to identify novel genes and pathways involved in the acquisition of new genomic changes in MM patients.We first developed a gene signature correlating with the genomic instability and survival in myeloma patients. To identify genes whose expression correlates with genomic instability, we used a myeloma patient dataset (gse26863, n=246) which had both the gene expression and CGH array-based copy number information for each patient. Genomic instability in each patient was determined by counting the total number of amplification and/or deletion events; an event was defined as a change in ≥3 and/or 5 consecutive probes. Genes whose elevated expression correlated with increased genomic instability in gse26863 dataset were tested for correlation with survival in two other datasets (IFM70, n=170; gse24080; n=559). Elevated expression of 287 genes correlated with poor overall (OS; P < 0.0001) as well as event free survival (EFS; P < 0.0001) in both these datasets. A pathway analysis identified cell cycle, chromosome maintenance, repair and replication as major pathways (P values < 0.00001). We confirmed their vaidity with other published genomic instability signatures. Of all the genes in our signature, 70% were common with Mitotic Chromosomal Instability signature (Sc Transl Med, 5 (181), 181ra50.[doi:17, 2013], 49% were present with published Chromosomal Instability Signature (CIN70; Nature Genetics, 38: 9, 2006) and 90% common with Chromosomal Instability Signature (CIN20; a subset of CIN70; Nature Genetics, 38: 9, 2006). We investigated the relevance of these genes with genomic instability, using a functional (HR activity) high throughput siRNA screen. Interestingly, >40% of the genes in our signature belonged to homologous recombination as indicated by our functional screen. We, therefore, initiated a functional siRNA screen of 124 genes to evaluate their impact on HR and nuclease activities. Based on the results, we next selected three genes for detailed evaluation. These included TTK (a dual specificity protein kinase), TPX2 (a spindle assembly factor required for normal assembly of mitotic spindles and microtubules) and USP21 (a deubiquitinase of histone H2A involved in epigenetic transcriptional repression). We suppressed these genes in MM cells using shRNA and confirmed that their suppression significantly reduces HR activity in MM cell lines. Suppression of USP21 was also associated with loss of RAD51 recombinase expression, suggesting its role in RAD51 stability. To monitor the impact of elevated expression of these genes on genomic integrity, we investigated knockdown cells for gH2AX levels using Western blotting. Knockdown of all 3 genes reduced gH2AX levels, indicating inhibition of spontaneous DNA breaks.In summary, our data has identified TTK, TPX2 and USP21 as novel genes involved in dysregulation of HR and genomic integrity in MM cells with potential impact on our understanding of the disease progression. DisclosuresNo relevant conflicts of interest to declare.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

ABSTRACTv‐Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v‐Src‐mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities‐mediated chromosome instability. v‐Src directly phosphorylates Tyr‐15 of cyclin‐dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v‐Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v‐Src restores cancer cell viability reduced by various microtubule‐targeting agents (MTAs), although v‐Src does not alter cytotoxicity of DNA‐damaging anticancer drugs. v‐Src causes mitotic slippage of MTAs‐treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v‐Src also restores cell viability reduced by a polo‐like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v‐Src. These results suggest that the v‐Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src‐induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Atpase Family AAA Domain-Containing Protein 2 (ATAD2) As a Novel Target in Multiple Myeloma

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P&amp;lt;0.0005 for both EFS and OS). We have begun to validate these genes for impact on genomic instability and on MM cell growth and survival. Here, we present the functional validation of AAA domain containing protein 2 (ATAD2), which is one of the top-most genes in our 30 gene signature in MM and has also been part of a chromosomal instability signature representing six different cancer types (Nature Genetics, 38: 9, 2006) and a mitotic chromosomal instability signature identified in breast cancer [Sci Transl Med, 2013]. Using The Cancer Genome Atlas data we have also correlated elevated ATAD2 expression with poor OS in pulmonary and pancreatic cancers (P&amp;lt;0.02). ATAD2 is a member of the AAA ATPase family of proteins containing two conserved ATPase domains and a bromodomain. Bromodomain containing proteins are involved in the regulation of gene expression and are frequently dysregulated in MM as well as other cancers. ATAD2 bromodomain selectively recognizes acetylated histone 4 (acetylated at K5 and K12) and has been shown to regulate many cellular processes including cell proliferation. ATAD2 has been shown to interact with and stimulate transcriptional activity of MYC and has also been shown to contribute to invasion and migration in several cancers. We have confirmed elevated ATAD2 expression relative to normal PBMC by Western blotting in all twelve myeloma cell lines tested. To further delineate its role in MM, we evaluated the impact of its knockdown on various parameters of growth and genome maintenance using shRNAs. Relative to control shRNA, knockdown with multiple different shRNAs resulted in near complete cell death in 3 MM cell lines (JJN3, H929 and RPMI) in five days. Reduced cell viability was accompanied by increased apoptosis as seen by annexin V/PI staining. Relative to control, ATAD2 knockdown in RPMI cells led to increase in the apoptotic cell fraction by 56% and in H929 cells by 42%. To investigate genomic impact of elevated ATAD2 expression, the live cell fraction from control and ATAD2-knockdown cells was evaluated, right after selection, for impact on the expression of γ-H2AX (a DNA break marker), pRPA32 (a marker of DNA end resection, a distinct step in the initiation of homologous recombination; HR) and recombinase RAD51 (a key player in HR). ATAD2-knockdown in H929 cells inhibited spontaneous DNA breaks, DNA end resection as well as RAD51 expression, suggesting that elevated ATAD2 contributes to increased spontaneous DNA damage and HR activity observed in MM cells. To further confirm its impact on genome stability, the live cell fraction from control and knockdown cells was evaluated for micronuclei (a marker of genomic instability). Relative to control shRNA-transduced cells, knockdown of ATAD2 cells reduced micronuclei by 58% in H929 and 53% in JJN3 cells. In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma.

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Guarding the Genome: CENP-A-Chromatin in Health and Cancer.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis.

FAT10 expression is highly up-regulated by pro-inflammatory cytokines IFNγ and TNFα in all cell types and tissues. Increased FAT10 expression may induce increasing mitotic non-disjunction and chromosome instability, leading to tumorigenesis. In this review, we summarized others’ and our work on FAT10 expression in liver biopsy samples from patients with alcoholic hepatitis (AH). FAT10 is essential to maintain the function of liver cell protein quality control and Mallory–Denk body (MDB) formation. FAT10 overexpression in AH leads to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10-/- mice. FAT10 causes the proteins’ accumulation, overexpression, and forming MDBs through modulating 26s proteasome’s proteases. The pathway that increases FAT10 expression includes TNFα/IFNγ and the interferon sequence response element (ISRE), followed by NFκB and STAT3, which were all up-regulated in AH. FAT10 was only reported in human and mouse specimens but plays critical role for the development of alcoholic hepatitis. Flavanone derivatives of milk thistle inhibit TNFα/IFNγ, NFκB, and STAT3, then inhibit the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα-response genes. Studies on Silibinin and other milk thistle derivatives to treat AH confirms that overexpressed FAT10 is the major key molecule in these networks.

MYC Dysregulates Mitosis, Revealing Cancer Vulnerabilities

Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

Aqueous extract of bulbus Fritillaria cirrhosa induces cytokinesis failure by blocking furrow ingression in human colon epithelial NCM460 cells

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.

Aurora kinase B-phosphorylated HP1α functions in chromosomal instability

ABSTRACTHeterochromatin Protein 1 α (HP1α) associates with members of the chromosome passenger complex (CPC) during mitosis, at centromeres where it is required for full Aurora Kinase B (AURKB) activity. Conversely, recent reports have identified AURKB as the major kinase responsible for phosphorylation of HP1α at Serine 92 (S92) during mitosis. Thus, the current study was designed to better understand the functional role of this posttranslationally modified form of HP1α. We find that S92-phosphorylated HP1α is generated in cells at early prophase, localizes to centromeres, and associates with regulators of chromosome stability, such as Inner Centromere Protein, INCENP. In mouse embryonic fibroblasts, HP1α knockout alone or reconstituted with a non-phosphorylatable (S92A) HP1α mutant results in mitotic chromosomal instability characterized by the formation of anaphase/telophase chromatin bridges and micronuclei. These effects are rescued by exogenous expression of wild type HP1α or a phosphomimetic (S92D) variant. Thus, the results from the current study extend our knowledge of the role of HP1α in chromosomal stability during mitosis.

MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis.

SummaryIn mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.

Depletion of Limiting rDNA Structural Complexes Triggers Chromosomal Instability and Replicative Aging of Saccharomyces cerevisiae.

Sir2 is a highly conserved NAD+-dependent histone deacetylase that functions in heterochromatin formation and promotes replicative life span (RLS) in the budding yeast, Saccharomyces cerevisiae Within the yeast rDNA locus, Sir2 is required for efficient cohesin recruitment and maintaining the stability of the tandem array. In addition to the previously reported depletion of Sir2 in replicatively aged cells, we discovered that subunits of the Sir2-containing complexes silent information regulator (SIR) and regulator of nucleolar silencing and telophase (RENT) were depleted. Several other rDNA structural protein complexes also exhibited age-related depletion, most notably the cohesin complex. We hypothesized that mitotic chromosome instability (CIN) due to cohesin depletion could be a driver of replicative aging. Chromatin immunoprecipitation assays of the residual cohesin (Mcd1-Myc) in moderately aged cells showed strong depletion from the rDNA and initial redistribution to the point centromeres, which was then lost in older cells. Despite the shift in cohesin distribution, sister chromatid cohesion was partially attenuated in aged cells and the frequency of chromosome loss was increased. This age-induced CIN was exacerbated in strains lacking Sir2 and its paralog, Hst1, but suppressed in strains that stabilize the rDNA array due to deletion of FOB1 or through caloric restriction. Furthermore, ectopic expression of MCD1 from a doxycycline-inducible promoter was sufficient to suppress rDNA instability in aged cells and to extend RLS. Taken together, we conclude that age-induced depletion of cohesin and multiple other nucleolar chromatin factors destabilize the rDNA locus, which then results in general CIN and aneuploidy that shortens RLS.

Therapeutic Targets for Adrenocortical Carcinoma in the Genomics Era

Adrenocortical carcinoma (ACC) is a rare and often fatal cancer, affecting ~1 person per million per year worldwide. Approximately 75% of patients with ACC eventually develop metastases and progress on the few available standard-of-care medical therapies, highlighting an incredible need for an improved understanding of the molecular biology of this disease. Although it has long been known that ACC is characterized by certain histological and genetic features (e.g., high mitotic activity, chromosomal instability, and overexpression of IGF2), only in the last two decades of genomics has the molecular landscape of ACC been more thoroughly characterized. In this review, we describe the findings of historical genetics and recent genomics studies on ACC and discuss how underlying concepts emerging from these studies contribute to the current model of critical pathways for adrenocortical carcinogenesis. Integrative synthesis across these studies reveals that ACC consists of three distinct molecular subtypes with divergent clinical outcomes and implicates differential regulation of Wnt signaling, cell cycle, DNA methylation, immune biology, and steroidogenesis in ACC biology. These cellular programs are pharmacologically targetable and may enable the development of therapeutic strategies to improve outcomes for patients facing this devastating disease.

Investigating the Promoter of FAT10 Gene in HCC Patients.

FAT10, which is also known as diubiquitin, has been implicated to play important roles in immune regulation and tumorigenesis. Its expression is up-regulated in the tumors of Hepatocellular Carcinoma (HCC) and other cancer patients. High levels of FAT10 in cells have been shown to result in increased mitotic non-disjunction and chromosome instability, leading to tumorigenesis. To evaluate whether the aberrant up-regulation of the FAT10 gene in the tumors of HCC patients is due to mutations or the aberrant methylation of CG dinucleotides at the FAT10 promoter, sequencing and methylation-specific sequencing of the promoter of FAT10 was performed. No mutations were found that could explain the differential expression of FAT10 between the tumor and non-tumorous tissues of HCC patients. However, six single nucleotide polymorphisms (SNPs), including one that has not been previously reported, were identified at the promoter of the FAT10 gene. Different haplotypes of these SNPs were found to significantly mediate different FAT10 promoter activities. Consistent with the experimental observation, differential FAT10 expression in the tumors of HCC patients carrying haplotype 1 was generally higher than those carrying haplotype II. Notably, the methylation status of this promoter was found to correlate with FAT10 expression levels. Hence, the aberrant overexpression of the FAT10 gene in the tumors of HCC patients is likely due to aberrant methylation, rather than mutations at the FAT10 promoter.

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.