Induction Of Chromosomal Instability Research Articles

A whale of a tale: whale cells evade the driving mechanism for hexavalent chromium-induced chromosome instability.

Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium [Cr(VI)] carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.

Molecular subtypes based on centrosome-related genes can predict prognosis and therapeutic responsiveness in patients with low-grade gliomas.

Abnormalities in centrosome regulatory genes can induce chromosome instability, cell differentiation errors, and tumorigenesis. However, a limited number of comprehensive analyses of centrosome-related genes have been performed in low-grade gliomas (LGG). LGG data were extracted from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. The ConsensusClusterPlus" R package was used for unsupervised clustering. We constructed a centrosome-related genes (CRGs) signature using a random forest model, lasso Cox model, and multivariate Cox model, and quantified the centrosome-related risk score (centS). The prognostic prediction efficacy of centS was evaluated using a Receiver Operating Characteristic (ROC) curve. Immune cell infiltration and genomic mutational landscapes were evaluated using the ESTIMATE algorithm, single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm, and "maftools" R package, respectively. Differences in clinical features, isocitrate dehydrogenase (IDH) mutation, 1p19q codeletion, O6-methylguanine-DNA methyltransferase promoter (MGMTp) methylation, and response to antitumor therapy between the high- and low-centS groups were explored. "pRRophetic" R packages were used for temozolomide (TMZ) sensitivity analysis. qRT-PCR verified the differential expression of the centrosomal gene team, the core of which is CEP135, between LGG cells and normal cells. Two distinct CRG-based clusters were identified using consensus unsupervised clustering analysis. The prognosis, biological characteristics, and immune cell infiltration of the two clusters differed significantly. A well-performing centS signature was developed to predict the prognosis of patients with LGG based on 12 potential CRGs. We found that patients in the high-centS group showed poorer prognosis and lower proportion of IDH mutation and 1p19q codeletion compared to those in the low-centS group. Furthermore, patients in the high-centS group showed higher sensitivity to TMZ, higher tumor mutation burden, and immune cell infiltration. Finally, we identified a centrosomal gene team whose core was CEP135, and verified their differential expression between LGG cells and normal glial cells. Our findings reveal a novel centrosome-related signature for predicting the prognosis and therapeutic responsiveness of patients with LGG. This may be helpful for the accurate clinical treatment of LGG.

MiR-186 induces tetraploidy in arsenic exposed human keratinocytes

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis.

HIV Infection, Chromosome Instability, and Micronucleus Formation.

Genome integrity is critical for proper cell functioning, and chromosome instability can lead to age-related diseases, including cancer and neurodegenerative disorders. Chromosome instability is caused by multiple factors, including replication stress, chromosome missegregation, exposure to pollutants, and viral infections. Although many studies have investigated the effects of environmental or lifestyle genotoxins on chromosomal integrity, information on the effects of viral infections on micronucleus formation and other chromosomal aberrations is still limited. Currently, HIV infection is considered a chronic disease treatable by antiretroviral therapy (ART). However, HIV-infected individuals still face important health problems, such as chronic inflammation and age-related diseases. In this context, this article reviews studies that have evaluated genomic instability using micronucleus assays in the context of HIV infection. In brief, HIV can induce chromosome instability directly through the interaction of HIV proteins with host DNA and indirectly through chronic inflammation or as a result of ART use. Connections between HIV infection, immunosenescence and age-related disease are discussed in this article. The monitoring of HIV-infected individuals should consider the increased risk of chromosome instability, and lifestyle interventions, such as reduced exposure to genotoxins and an antioxidant-rich diet, should be considered. Therapies to reduce chronic inflammation in HIV infection are needed.

CEP63 upregulates YAP1 to promote colorectal cancer progression through stabilizing RNA binding protein FXR1.

Abnormal regulation of centrosome components can induce chromosome instability and tumorigenesis. Centrosomal protein 63 (CEP63) is a vital member for assembling centrosome. Yet, the involvement of CEP63 in cancer pathogenesis remains unclear. Here we identify CEP63 as an important mediator for RNA-binding proteins (RBPs) to facilitate regulation on their RNA targets in colorectal cancer (CRC). We demonstrate that CEP63 protein is upregulated in a large cohort of colorectal cancer tissues and predicts poor prognosis, and USP36 is identified for stabilizing CEP63 by enhancing its K48-dependent deubiquitination. CEP63 overexpression promotes the proliferation and tumor growth of CRC cells in vitro and in vivo. Furthermore, we find that CEP63 can promote cancer stem-like cell properties by enhancing YAP1 expression through binding with and inhibiting the K63-ubiquitylation degradation of RBP FXR1 in CRC cells. Importantly, we further verify that the KH domain of FXR1 is necessary for the interaction between CEP63 and FXR1. Moreover, microtube motor proteins can form a complex with CEP63 and FXR1 to mediate the regulation of FXR1 on RNA targets. Additionally, we also confirm that CEP63 can bind and regulate multiple RBPs. In conclusion, our findings unveil an unrecognized CEP63/RBPs/RNA axis that CEP63 may perform as an adapter facilitating the formation of RBPs complex to regulate RNA progression and discover the role of CEP63 involved in signal transduction and RNA regulation, providing potential therapeutic target for CRC patients.

Abstract 705: Inhibition of H3.3 S31 phosphorylation by the pediatric glioma driver mutation, H3.3 K27M, results in chromosomal instability, loss of p53 regulation, and tumorigenesis

Abstract The heterozygous H3.3 K27M mutation is found in the majority of pediatric High-Grade Glioma’s (pHGG). H3.3 K27M is thought to promote tumor formation through altered epigenetic gene regulation. However, it is unclear if epigenetic reprogramming alone is sufficient for glioma formation. H3.3 contains a unique Serine at position 31 which is phosphorylated in early mitosis (where it functions in chromosome segregation) and again in late M/early G1 when a mitotic error has occurred (triggering p53 expression). We found that pHGG cell lines harboring H3.3 K27M have significantly reduced S31 phosphorylation as compared to both pHGG and non-transformed H3.3 WT cells. When compared to WT H3.3 in an in vitro kinase assay, H3.3 K27M exhibits an ~60% reduction in S31 phosphorylation by Chk1, the mitotic S31 kinase. In normal diploid cells, expression of K27M or non-phosphorylatable S31A mutant significantly increased chromosome missegregation. Yet expressing a phosphomimetic double mutant (K27M/S31E) did not significantly alter the rate of chromosome mis-segregation compared to WT. Furthermore, patient-derived pHGG lines harboring K27M have significantly higher rates of chromosome mis-segregation compared to an H3.3 WT pHGG line or H3.3 WT non-transformed human cells. We used CRISPR gene editing to remove the H3.3 K27M allele or replace it with WT H3.3. In both cases, loss of K27M elevated pS31 levels and reduced chromosome mis-segregations. Yet, when K27M was replaced with S31A, the mis-segregation rate remained similar to the parental K27M cells. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. To determine if the induction of chromosomal instability would alter tumor formation, we expressed H3.3 WT, K27M or S31A in combination with PDGFβ in a glioma mouse model. Expression of WT H3.3 failed to generate high-grade tumors. But 66% of mice expressing K27M and 93% of those expressing H3.3 S31A developed diffuse high-grade brain tumors by 100 days. Our results suggest that loss of phospho-S31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest - thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming. Citation Format: Charles Day, Florina Grigore, Alyssa Langfald, David J. Daniels, James Robinson, Edward Hinchcliffe. Inhibition of H3.3 S31 phosphorylation by the pediatric glioma driver mutation, H3.3 K27M, results in chromosomal instability, loss of p53 regulation, and tumorigenesis [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 705.

HGG-30. H3.3 G34R/V mutations disrupt H3.3 mitotic phosphorylation leading to high-grade glioma formation through the induction of chromosomal instability

Pediatric high-grade gliomas (pHGG) are among the most lethal of all human cancers. Histone H3.3 G34R/V mutations are an early event in these tumors and show reduced H3.3 K36 trimethylation; implicating epigenetic dysregulation in tumorigenesis. Here we present evidence that H3.3 G34R/V mutations promote tumor formation via the induction of chromosomal instability (CIN). Pericentromeric H3.3 is phosphorylated at S31 by Chk1 during mitosis. We observed that the H3.3 G34R mutation reduced Chk1 phosphorylation of H3.3 S31 by >90% in vitro. Furthermore, H3.3 G34 mutant cells have reduced pericentromeric H3.3 S31 phosphorylation in mitosis compared to WT H3.3 cell lines. H3.3 G34 mutant pHGG cells also have significantly elevated rates of CIN as compared to H3.3 WT cells. Overexpression of H3.3 G34R, G34V or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN, but H3.3 K36M overexpression had no effect on chromosome segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to induce CIN in normal, diploid cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation.

Abstract LB119: VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient

Abstract Spindle assembly checkpoint (SAC) prevents mis-segregation of chromosome and allows accurate division in mitosis. Mps1 (also known as TTK) is one of the essential kinases for recruitment of SAC proteins after chromosome segregation. Failure of SAC induces chromosome instability, mitotic catastrophe, and ultimately apoptotic cell death and Mps1 is induced or activated in rapidly dividing tumor cells. Therefore, Mps1 has been considered an attractive anti-cancer drug target. VRN081569 is a potent type I kinase inhibitor of Mps1, which stabilizes Mps1 kinase domain in a closed conformation by forming a tight interface with its kinase domain. This structural stabilization appears to increase cellular potency for the induction of aneuploidy and apoptosis (~ 10 nM or less GI50 value). The loss of retinoblastoma tumor suppressor gene (Rb1), which is prevalent in advanced cancers, dysregulates cell cycle and promotes cell division. In particular, the resistance to CDK4/6 inhibitors has been reportedly mediated by loss of the Rb1 gene. We found that Rb1-negative cells express higher level of Mps1 than Rb1-positive cells and VRN081569 efficiently induce apoptosis of Rb1-negative Triple Negative Breast Cancer (TNBC) cell line MDA-MB-468 than Rb1-positive TNBC MDA-MB-231. Especially we confirmed that VRN081569 induces caspase 3/7 activity faster in Rb1-negative cells (~50-fold EC50 of caspase 3/7 activity compared to Rb1-positive cells), and cell cycle analysis has shown that our compound induces a dose-dependent dysregulation of mitosis, resulting in a decrease in the frequency of the G0/G1 phase, and also induces aneuploidy and apoptosis. Finally, we confirmed a combination effects with anti-microtubule agents BCL-2 inhibitor and PARP-1 inhibitor, suggesting that modulation of two different cellular processes may overcome the lack of single agent activity for TNBC. Citation Format: Hong Ryul Jung, Yeonsil Kim, Haelee Kim, Ji-Hye Kwon, Dong-hyuk Seo, Jung Beom Son, Nam Doo Kim, Daekwon Kim, Sunghwan Kim. VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient [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 LB119.

HGG-31. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

Reduced USP22 Expression Impairs Mitotic Removal of H2B Monoubiquitination, Alters Chromatin Compaction and Induces Chromosome Instability That May Promote Oncogenesis.

Simple SummaryChromosome instability (CIN) promotes cancer initiation and progression, but its genetic origins remain largely unknown. As aberrant mitotic processes such as chromatin compaction defects can induce CIN, characterizing the molecular determinants of mitotic fidelity may reveal novel CIN genes. In vitro, monoubiquitination of histone H2B at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted in early mitosis. USP22 is a major deubiquitinating enzyme catalyzing H2Bub1 removal in interphase and may also be responsible for H2Bub1 depletion in mitosis. To assess whether mitotic removal of H2Bub1 is required for mitotic fidelity, we employ complementary genetic and single-cell imaging microscopy approaches to assess the impact reduced USP22 expression has on H2Bub1 abundance, chromatin compaction and chromosome stability. We show that USP22 deficiency impairs H2Bub1 removal and induces chromatin compaction defects. Further, we identify USP22 as a novel CIN gene, indicating that USP22 deletions in tumors may drive CIN and contribute to oncogenesis.Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.

Particulate Hexavalent Chromium Inhibits E2F1 Leading to Reduced RAD51 Nuclear Foci Formation in Human Lung Cells

Abstract Lung cancer is the leading cause of cancer death; however, the mechanisms of lung carcinogens are poorly understood. Metals, including hexavalent chromium [Cr(VI)], induce chromosome instability, an early event in lung cancer. Failure of homologous recombination repair is a key mechanism for chromosome instability. Particulate Cr(VI) causes DNA double-strand breaks and prolonged exposure impairs homologous recombination targeting a key effector protein in this pathway, RAD51. Reduced RAD51 protein is a key endpoint of particulate Cr(VI) exposure. It is currently unknown how Cr(VI) reduces RAD51 protein. E2F1 is the predominant transcription factor for RAD51. This study sought to identify if E2F1 modulates the RAD51 response to particulate Cr(VI). Particulate Cr(VI) reduced RAD51 protein and mRNA levels but had a minimal effect on RAD51 half-life. E2F1 protein and mRNA were also inhibited by particulate Cr(VI) exposure. To connect these two outcomes, we tested if modulating E2F1 affects RAD51 outcomes after particulate Cr(VI) exposure. E2F1 knockdown inhibited RAD51 nuclear foci formation after acute particulate Cr(VI) exposure. These data indicate reduced RAD51 protein levels after prolonged particulate Cr(VI) exposure are predominantly due to inhibited expression. Particulate Cr(VI) also inhibits E2F1 expression. However, although loss of E2F1 does not modulate RAD51 expression after particulate Cr(VI) exposure, RAD51 nuclear foci formation is inhibited. These findings suggest E2F1 is important for RAD51 localization to double-strand breaks, but not expression after particulate Cr(VI) exposure in human lung cells.

Reduced RBX1 expression induces chromosome instability and promotes cellular transformation in high-grade serous ovarian cancer precursor cells.

Despite high-grade serous ovarian cancer (HGSOC) being the most common and lethal gynecological cancer in women, the early etiological events driving disease development remain largely unknown. Emerging evidence now suggests that chromosome instability (CIN; ongoing changes in chromosome numbers) may play a central role in the development and progression of HGSOC. Importantly, genomic amplification of the Cyclin E1 gene (CCNE1) contributes to HGSOC pathogenesis in ~20% of patients, while Cyclin E1 overexpression induces CIN in model systems. Cyclin E1 levels are normally regulated by the SCF (SKP1-CUL1-FBOX) complex, an E3 ubiquitin ligase that includes RBX1 as a core component. Interestingly, RBX1 is heterozygously lost in ~80% of HGSOC cases and reduced expression corresponds with worse outcomes, suggesting it may be a pathogenic event. Using both short (siRNA) and long (CRISPR/Cas9) term approaches, we show that reduced RBX1 expression corresponds with significant increases in CIN phenotypes in fallopian tube secretory epithelial cells, a cellular precursor of HGSOC. Moreover, reduced RBX1 expression corresponds with increased Cyclin E1 levels and anchorage-independent growth. Collectively, these data identify RBX1 as a novel CIN gene with pathogenic implications for HGSOC.

Chromosome instability induced by a single defined sister chromatid fusion.

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Abstract 5814: Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription

Abstract A better understanding of the precise molecular mechanisms that control cell proliferation is crucial for the identification of novel cancer therapeutic targets. A critical molecule in this process is the Cyclin-Dependent Kinase 4 (CDK4), which regulates key aspects of the G1 to S phase transition through phosphorylation of the retinoblastoma (Rb) family of proteins. Since dysregulation of this cell cycle mechanism is a hallmark of most cancers, drugs have been developed to inhibit the kinase activity of CDK4. However, such drugs have been met with variable degrees of success and, in some cases, little efficacy. Research performed in the last few years has suggested that CDK4 have alternative targets other than the Rb proteins. Consistent with this idea, we found that CDK4 plays additional roles not related to the canonical function in the CDK-Rb axis. Here, we show that CDK4 remains attached to the chromatin fraction of Balb/MK2 keratinocytes upon the extraction of the soluble components (nucleoplasm and cytoplasm). Chromatin-immunoprecipitation (ChIP) analysis shows that CDK4 occupies the regulatory sites of genes associated with chromosomal segregation, such as Aurkb (Aurora B) and Cenpp (Centromere Protein P). Moreover, gain- and loss-of-function experiments showed that the AurkB and Cenpp promoters are regulated through CDK4 binding. In line with these findings, studies in skin carcinogenesis revealed that transgenic expression of CDK4 in mouse epidermis induces chromosome instability (CIN) and centrosome amplification (CA), two events linked to overexpression of Aurora B. Moreover, Aurora-B has a crucial role in chromosome bi-orientation and the spindle-assembly checkpoint, whereas Cenpp is required for proper kinetochore function. Remarkably, a persistent dysregulation of Aurora B has been reported in numerous human cancers. Thus, our results suggest that CDK4 plays a role in the maintenance of chromosomal stability, implying that additional functions could be inhibited by therapeutically targeting CDK4. Our conclusions are supported by previous reports showing that pharmacological inhibition of CDK4 led to delayed progression from G2 to mitosis in Rb-positive and as well as Rb-negative cells. Citation Format: Sung Hyun Lee, Liliana R. Rodriguez, Paula L. Miliani de Marval, Marcelo L. Rodriguez-Puebla. Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5814.

Clinical Implications of the Associations Between Intestinal Microbiome and Colorectal Cancer Progression.

Intestinal microbiome influences host immunity and several diseases, including cancer, in their areas of colonization. Microbial dysbiosis and over-colonization of specific microbes within the colorectal mucosa can impact the progress of carcinogenesis. Investigations initially focused on the mechanisms by which the intestinal microbiome initiates or promotes the development of colorectal cancer, including DNA damage, induction of chromosomal instability, and regulation of host immune responses. Some studies on the clinicopathological features have reported that specific strains present at high abundance are associated with advanced stage and positive lymph nodes in colorectal cancer. In this context, we reviewed the relationship between the intestinal microbiome and the clinical features (patient age, disease staging, prognosis, etc.) of patients with colorectal cancer, and evaluated the potential pathogenesis caused by the intestinal microbiome in disease progress. This article assessed whether changes in distinct species or strains occur during the period of cancer advancement. Overall, age grouping does not bring about significant differences in the constitution of microbiome. The disease stages show their distinct distribution in some species and strains. Oncogenic species are generally enriched in patients with poor prognosis, including low infiltration of CD3+ T cells, poor differentiation, widespread invasion, high microsatellite instability, CpG island methylator phenotype, BRAF mutation, short overall survival, and disease-free survival. The implications of those changes we discussed may assist in comprehensive understanding of the tumorigenesis of colorectal cancer from a microbiological perspective, finding potential biomarkers for colorectal cancer.

The cGAS/STING/TBK1/IRF3 innate immunity pathway maintains chromosomal stability through regulation of p21 levels

Chromosomal instability (CIN) in cancer cells has been reported to activate the cGAS–STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, adverse effects of the cGAS/STING pathway as they relate to CIN have not yet been investigated. We addressed this issue using knockdown and add-back approaches to analyze each component of the cGAS/STING/TBK1/IRF3 pathway, and we monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in increased micronuclei formation and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK-binding kinase-1), or IRF3 (interferon regulatory factor-3) also resulted in increased micronuclei formation. Moreover, transfection with cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1-, and IRF3-knockdown cells, which was accompanied by the precocious G2/M transition of cells and the enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated the precocious G2/M transition, indicating that the decrease in p21 and the subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN through disruption of cGAS/STING signaling.

Reduced SKP1 Expression Induces Chromosome Instability through Aberrant Cyclin E1 Protein Turnover.

Chromosome instability (CIN), or progressive changes in chromosome numbers, is an enabling feature of many cancers; however, the mechanisms giving rise to CIN remain poorly understood. To expand our mechanistic understanding of the molecular determinants of CIN in humans, we employed a cross-species approach to identify 164 human candidates to screen. Using quantitative imaging microscopy (QuantIM), we show that silencing 148 genes resulted in significant changes in CIN-associated phenotypes in two distinct cellular contexts. Ten genes were prioritized for validation based on cancer patient datasets revealing frequent gene copy number losses and associations with worse patient outcomes. QuantIM determined silencing of each gene-induced CIN, identifying novel roles for each as chromosome stability genes. SKP1 was selected for in-depth analyses as it forms part of SCF (SKP1, CUL1, FBox) complex, an E3 ubiquitin ligase that targets proteins for proteolytic degradation. Remarkably, SKP1 silencing induced increases in replication stress, DNA double strand breaks and chromothriptic events that were ascribed to aberrant increases in Cyclin E1 levels arising from reduced SKP1 expression. Collectively, these data reveal a high degree of evolutionary conservation between human and budding yeast CIN genes and further identify aberrant mechanisms associated with increases in chromothriptic events.

Reduced Expression of Genes Regulating Cohesion Induces Chromosome Instability that May Promote Cancer and Impact Patient Outcomes

Chromosome instability (CIN), or continual changes in chromosome complements, is an enabling feature of cancer; however, the molecular determinants of CIN remain largely unknown. Emerging data now suggest that aberrant sister chromatid cohesion may induce CIN and contribute to cancer. To explore this possibility, we employed clinical and fundamental approaches to systematically assess the impact reduced cohesion gene expression has on CIN and cancer. Ten genes encoding critical functions in cohesion were evaluated and remarkably, each exhibits copy number losses in 12 common cancer types, and reduced expression is associated with worse patient survival. To gain mechanistic insight, we combined siRNA-based silencing with single cell quantitative imaging microscopy to comprehensively assess the impact reduced expression has on CIN in two karyotypically stable cell lines. We show that reduced expression induces CIN phenotypes, namely increases in micronucleus formation and nuclear areas. Subsequent direct tests involving a subset of prioritized genes also revealed significant changes in chromosome numbers with corresponding increases in moderate and severe cohesion defects within mitotic chromosome spreads. Collectively, our clinical and fundamental findings implicate reduced sister chromatid cohesion, resulting from gene copy number losses, as a key pathogenic event in the development and progression of many cancer types.

Suppression of tumor antigen presentation during aneuploid tumor evolution contributes to immune evasion

ABSTRACTAnti-tumor immune responses impede tumor formation, and cancers have evolved many mechanisms of immune evasion. Confirming earlier findings, we show that human tumors with high chromosomal instability (CIN+) are significantly less immunogenic, as judged by tumor lymphocyte infiltration, compared to those with more stable genomes (CIN-). This finding is paradoxical, as genomic instability is expected to evoke an innate immune response. Importantly, CIN+ tumors and cell lines exhibited suppressed expression of proteins involved in MHC class I antigen presentation at least partly due to DNA hypermethylation of the corresponding genes. Using a mouse model of the in vivo evolution of aneuploid tumors, we found that the induction of chromosomal instability in tumor cells is highly immunogenic due to the activation of the STING/TBK1 pathway and consequent increased interferon signaling and antigen presentation. However, tumors evolving under immune pressure suppress the STING/TBK1 and antigen presentation pathways and evade anti-tumor immune responses. In contrast, CIN+ tumors that develop under low immune pressure in both humans and mice retain efficient MHC class I antigen presentation and immunogenicity. Altogether, this study identifies an important mechanism of immune evasion in chromosomally unstable tumors.

Chromosome instability caused by mutations in the genes involved in transcription and splicing

ABSTRACTMutations in molecules involved in transcription and splicing can cause chromosome instability such as sister chromatid exchanges. We isolated and characterized responsible genes from mammalian temperature-sensitive mutant cells showing chromosome instability. A mutation in the largest subunit of RNA polymerase II affected DNA synthesis in S phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges. The yeast mutant harboring a homologous mutation showed very similar phenotype to that of the mammalian mutant. A mutation in Smu1, which is involved in splicing, also affected DNA synthesis in S and G2 phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges and chromosomal aberrations. These cells showed a connection between defects of RNA metabolism and induction of chromosome instability. Genome instability appeared to be caused by links between RNA metabolism and replication resulting in genomic recombination. RNA metabolism can be regarded as one possible driver of genome modification triggering genome evolution