Cell Cycle-dependent Expression Research Articles

Evidence for novel mechanisms that control cell-cycle entry and cell size.

Entry into the cell cycle in late G1 phase occurs only when sufficient growth has occurred. In budding yeast, a cyclin called Cln3 is thought to link cell-cycle entry to cell growth. Cln3 accumulates during growth in early G1 phase and eventually helps trigger expression of late G1 phase cyclins that drive cell-cycle entry. All current models for cell-cycle entry assume that expression of late G1 phase cyclins is initiated at the transcriptional level. Current models also assume that the sole function of Cln3 in cell-cycle entry is to promote transcription of late G1 phase cyclins, and that Cln3 works solely in G1 phase. Here, we show that cell cycle-dependent expression of the late G1 phase cyclin Cln2 does not require any functions of the CLN2 promoter. Moreover, Cln3 can influence accumulation of Cln2 protein via posttranscriptional mechanisms. Finally, we show that Cln3 has functions in mitosis that strongly influence cell size. Together, these discoveries reveal the existence of surprising new mechanisms that challenge current models for control of cell-cycle entry and cell size.

A transient increase of HIF-1α during the G1 phase (G1-HIF) ensures cell survival under nutritional stress

The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.

Regulation of the mitotic chromosome folding machines.

Over the last several years enormous progress has been made in identifying the molecular machines, including condensins and topoisomerases that fold mitotic chromosomes. The discovery that condensins generate chromatin loops through loop extrusion has revolutionized, and energized, the field of chromosome folding. To understand how these machines fold chromosomes with the appropriate dimensions, while disentangling sister chromatids, it needs to be determined how they are regulated and deployed. Here, we outline the current understanding of how these machines and factors are regulated through cell cycle dependent expression, chromatin localization, activation and inactivation through post-translational modifications, and through associations with each other, with other factors and with the chromatin template itself. There are still many open questions about how condensins and topoisomerases are regulated but given the pace of progress in the chromosome folding field, it seems likely that many of these will be answered in the years ahead.

Regulating the regulator: a survey of mechanisms from transcription to translation controlling expression of mammalian cell cycle kinase Aurora A.

Aurora Kinase A (AURKA) is a positive regulator of mitosis with a strict cell cycle-dependent expression pattern. Recently, novel oncogenic roles of AURKA have been uncovered that are independent of the kinase activity and act within multiple signalling pathways, including cell proliferation, survival and cancer stem cell phenotypes. For this, cellular abundance of AURKA protein is per se crucial and must be tightly fine-tuned. Indeed, AURKA is found overexpressed in different cancers, typically as a result of gene amplification or enhanced transcription. It has however become clear that impaired processing, decay and translation of AURKA mRNA can also offer the basis for altered AURKA levels. Accordingly, the involvement of gene expression mechanisms controlling AURKA expression in human diseases is increasingly recognized and calls for much more research. Here, we explore and create an integrated view of the molecular processes regulating AURKA expression at the level of transcription, post-transcription and translation, intercalating discussion on how impaired regulation underlies disease. Given that targeting AURKA levels might affect more functions compared to inhibiting the kinase activity, deeper understanding of its gene expression may aid the design of alternative and therapeutically more successful ways of suppressing the AURKA oncogene.

Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena.

Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images.

Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.

Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.

Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs—SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1—for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.

Zygote arrest 1 like (ZAR2) and cell cycle in breast cancer cells

ZAR2 is a novel RNA-binding C4 zinc finger containing protein, with cell cycle dependent differential expression and nuclear/ cytosolic localization. We observed that ZAR2 expression is relatively lower in triple negative breast cancer cells (TNBC) compared to non-TNBC and forced expression in TNBC cells inhibit their growth and invasiveness. Knocking down ZAR2 in non-TNBC cells increased their growth and invasiveness. Our studies showed that ZAR2, is inhibited by a c-Myc regulated miRNA, hsa-miR-130a-5p. Studies demonstrate that ZAR2 localizes in the RNA processing P-bodies and regulates the turnover of maternal WEE1 mRNA in developing oocytes. WEE1 is a tyrosine kinase, which regulates G2/M checkpoint in cell cycle, is over-expressed in various cancer types and is associated with resistance development to DNA-damaging agents. WEE1, is identified as a potential molecular target for TNBC. WEE1 inhibition is exclusively toxic for cancer cells and does not harm healthy cells. Major objective of the proposed research is to evaluate the potential of a novel protein ZAR2 as a modulator of G2-M checkpoint and therapeutic target in breast cancer. c-Myc mediated downregulation of ZAR2 expression via hsa-miR-130a-5p, results in increased levels of WEE1 protein in breast cancer cells, increased proliferation and faster progression through cell cycle.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Active and Passive Destabilization of G-Quadruplex DNA by the Telomere POT1-TPP1 Complex

Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.

CDK4 and CDK5 Inhibition Have Comparable Mild Hypothermia Effects in Preventing Drp1-Dependent Mitochondrial Fission and Neuron Death Induced by MPP.

Mild hypothermia has promising effects in the treatment of acute brain insults and also affects cell cycle progression. Mitochondrial dynamics, fusion and fission, are changed along with the cell cycle and disrupted in neurodegenerative diseases, including Parkinson's disease (PD). However, the effects of hypothermia on aberrant mitochondrial dynamics in PD remain unknown. We hypothesized that mild hypothermia protects neurons by regulating cell cycle-dependent protein expression and mitochondrial dynamics in a 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of PD. We found that the hypothermia treatment at 32°C prevented MPP+-induced neuron death; however, 32°C treatment itself also reduced cell viability. This reduction was associated with cell cycle arrest and downregulation of cyclin-dependent kinase 4 (CDK4) in proliferating human SK-N-SH neuroblastoma cells but upregulation in well-differentiated primary rat cortical neurons. In both types of neurons, hypothermia upregulated p27 (an endogenous inhibitor of CDKs) and p35 (CDK5 activator) protein expression. Treatment with hypothermia, or a selective CDK4 inhibitor, or roscovitine (CDK5 inhibitor) prevented MPP+-induced mitochondrial fission, upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1), and neuron death. In addition, overexpression of dominant negative mutant Drp1K38A improved MPP+-induced mitochondrial fission while overexpression of wild-type Drp1 blunted the prevention of mitochondrial fission by hypothermia as well as CDK4 inhibitor and roscovitine. These results elucidate that hypothermia may inhibit CDK4 and CDK5 activation by upregulating p27 and p35 expression to prevent Drp1-dependent mitochondrial fission and neuron loss after MPP+ treatment. CDK4 and CDK5 inhibition imitates the neuroprotective functions of hypothermia as a potential therapy for PD.

First person – Shivnarayan Dhuppar

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shivnarayan Dhuppar is first author on ‘Investigating cell cycle-dependent gene expression in the context of nuclear architecture at a single-allele resolution’, published in JCS. Shivnarayan is a postdoc in the lab of Dr Aprotim Mazumder at Tata Institute of Fundamental Research, Hyderabad, India, where he likes to think ‘how does it work?’, with the ‘it’ being anything from the biology of a cell to its functions and the physics behind them.

Investigating cell cycle-dependent gene expression in the context of nuclear architecture at single-allele resolution.

Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as the nuclear lamina, nuclear matrix or nucleoli. Recently, nuclear architecture has emerged as a major regulator of gene expression in mammalian cells. However, studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or gene expression within a population. In this report we present a method for combining 3D DNA fluorescence in situ hybridization (FISH) with single-molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine our method with imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of the cyclin-A2 (CCNA2) gene, which has known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position - which usually lies close to nuclear lamina - nor on the expression from other copies of the gene.This article has an associated First Person interview with the first author of the paper.

Abstract GMM-031: CLINICAL PATHOLOGIC EXPRESSION OF CELL CYCLE REGULATORY COMPLEXES IN HIGH GRADE SEROUS OVARIAN CARCINOMA

Abstract INTRODUCTION: Cell cycle control is an important determinant of cancer progression and treatment response. Two key transcriptional regulatory complexes, DREAM and MMB, ensure coordinated cell cycle dependent gene expression. Though these complexes contain the same protein core called MuvB, they have opposing functions. The DREAM (DP, RB-like, E2F, and MuvB) complex represses over 800 cell cycle genes in G0/G1 and the MMB (Myb-MuvB) complex promotes mitotic gene expression. High expression of B-Myb, an oncogenic transcription factor involved in the MMB complex, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. High B-Myb expression disrupts DREAM formation in human cell lines, resulting in increased proliferation. Previous analysis of TCGA data showed that MYBL2 (encoding B-Myb) undergoes gene copy number gain in 55% of high grade serous ovarian cancer (HGSOC) tumor samples and is associated with poor overall survival. We sought to validate these findings with clinical specimens and to investigate the role of DREAM- and MMB-regulated gene expression in HGSOC patient outcomes. METHODS: We used expression levels of DREAM-and MMB-controlled genes as a functional readout for the status of these opposing transcriptional regulators. This retrospective study utilized tissue bank surgical pathology and cytology samples taken from 52 HGSOC lesions. RT-qPCR gene expression analysis was correlated to clinical and pathologic findings. Demographic information, follow-up, treatment, and outcomes data (age, Stage, optimal debulking, platinum sensitivity, survival) was obtained by chart review. Analyses of TCGA datasets were conducted in parallel. RESULTS: RT-qPCR analysis of DREAM target genes (AURKA, KIF23, CCNB2, and FOXM1) revealed positive and significant correlations between MYBL2 and all genes tested: AURKA (ρ=0.4114, p<0.01), KIF23 (ρ=0.4953, p<0.001), CCNB2 (ρ=0.3278, p<0.05), and FOXM1 (ρ=0.5033, p<0.001). Stage at diagnosis, optimal debulking, platinum sensitivity and survival did not associate with high or low expression of target genes. High FOXM1 expression was associated with longer progression free survival (p<0.01). High CCNB2 (encoding cyclin B2) showed a trend (p=0.0643) with decreased overall survival, with a median time difference of 20 months between high (26 months) and low (46 months) groups. Analysis of TCGA HGSOC datasets revealed that DREAM and MMB target genes were significantly upregulated in the presence of high B-Myb expression (Fisher's exact test, p<0.01). The top 49 upregulated genes associated with high MYBL2 in HGSOC analysis have been previously annotated as DREAM target genes (χ2 with Yates correction p<0.001). CONCLUSIONS: Increased expression of selected cell cycle genes correlates to increased formation of MMB, and reduced DREAM assembly in HGSOC tissue. High expression of MYBL2 is associated with deregulated cell cycle gene expression programs in HGSOC. Larger scale studies would clarify the clinical prognostic value of the DREAM- and MMB-regulated gene expression. Citation Format: Audra N. Iness, Lisa Rubinsak, Jessica Chaoul, Mikhail Dozmorov, Cora Uram-Tuculescu, Larisa Litovchick, Sarah Temkin. CLINICAL PATHOLOGIC EXPRESSION OF CELL CYCLE REGULATORY COMPLEXES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-031.

RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21

CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific alternative splicing and contributes to the pathogenesis of myotonic dystrophy (DM), however the role of CELF6 in cancer cell proliferation is less appreciated. Here, we show that the expression of CELF6 is cell cycle regulated. The cell cycle-dependent expression of CELF6 is mediated through the ubiquitin-proteasome pathway, SCF-β-TrCP recognizes a nonphospho motif in CELF6 and regulates its proteasomal degradation. Overexpression or depletion of CELF6 modulates p21 gene expression. CELF6 binds to the 3′UTR of p21 transcript and increases its mRNA stability. Depletion of CELF6 promotes cell cycle progression, cell proliferation and colony formation whereas overexpression of CELF6 induces G1 phase arrest. The effect of CELF6 on cell proliferation is p53 and/or p21 dependent. Collectively, these data demonstrate that CELF6 might be a potential tumor suppressor, CELF6 regulates cell proliferation and cell cycle progression via modulating p21 stability.

Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression

The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.

Promoter-dependent nuclear RNA degradation ensures cell cycle-specific gene expression

Cell cycle progression depends on phase-specific gene expression. Here we show that the nuclear RNA degradation machinery plays a lead role in promoting cell cycle-dependent gene expression by triggering promoter-dependent co-transcriptional RNA degradation. Single molecule quantification of RNA abundance in different phases of the cell cycle indicates that relative curtailment of gene expression in certain phases is attained even when transcription is not completely inhibited. When nuclear ribonucleases are deleted, transcription of the Saccharomyces cerevisiae G1-specific axial budding gene AXL2 is detected throughout the cell cycle and its phase-specific expression is lost. Promoter replacement abolished cell cycle-dependent RNA degradation and rendered the RNA insensitive to the deletion of nuclear ribonucleases. Together the data reveal a model of gene regulation whereby RNA abundance is controlled by promoter-dependent induction of RNA degradation.

Kub5‐HeraRPRD1B regulates CDK1 expression through the DREAM complex

Triple Negative Breast Cancer (TNBC) is a subtype with more aggressive, less differentiated capacity and enhanced metastatic ability. Although TNBC tumors are more responsive to neoadjuvant chemotherapy compared to receptor‐positive tumors, development of resistance to cytotoxic chemotherapy and early relapse is more common. Due to the lack of effective therapies, understanding the underlying mechanisms of TNBC aggressiveness and poor prognosis is a major focus of research with particular interest in identifying new therapeutic targets.Kub5‐HeraRPRD1B (K‐H) is a novel factor involved in preserving genomic stability and regulates BRCA1 function by mediating the expression of CDK1, a master regulator of cell cycle, DNA replication, and DNA repair. This regulation has been reported in MDA‐MB‐231 cells, a model of TNBC. However, the underlying mechanism regulated CDK1 expression by K‐H is unknown. Here, we found that the CDK1 promoter is essential for the recruitment of K‐H. We also examined the interaction between K‐H and the complex for the regulation of cell cycle‐dependent gene expression, DREAM complex (dimerization partner, RB‐like, E2F and multi‐vulval class B complex) by immunoprecipitation. The interaction between K‐H and the DREAM complex is further validated using a Proximity ligation assay (PLA). Furthermore, a mutation in K‐H (R106A) results in the loss of an interaction between K‐H and the DREAM complex as well as K‐H and RNAPII, leading to decreased CDK1 levels. These results suggest that CDK1 expression is upregulated by K‐H and its complex formation with the DREAM complex and RNAPII. Thus, we hypothesize that K‐H modulates CDK1 expression through the recruitment of the DREAM complex to the CDK1 promoter region in a sequence‐specific manner. Our studies reveal a novel strategy for developing therapies that target the interaction between K‐H and the DREAM complex, potentially inhibiting the growth of cancer cells.Support or Funding InformationThis work is supported by NIH grant 1 R01 CA201489‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3364: RAI2 maintains genome stability of breast cancer cells

Abstract Genome instability is considered as an enabling hallmark of cancer formation and progression as well as a source of intratumoral heterogeneity. Retinoid acid-induced protein 2 (RAI2) was initially identified as a new metastasis-associated protein especially related with the presence of disseminated tumor cells in the bone marrow of breast cancer patients. In primary breast tumors low RAI2 gene expression correlates with mutant TP53 status and moreover, RAI2 depleted luminal breast cancer cell lines show decreased expression of key regulators of G2/M transition. We hypothesize that RAI2 has an influence on mitotic fidelity and is involved in maintaining genomic stability in breast cancer cell lines. To analyze consequences of RAI2 depletion on mitotic fidelity in luminal breast cancer cells, we followed mitotic cells by live cell imaging and analyzed chromosomal segregation by immune fluorescence staining and karyotyped RAI2 depleted cells. For analysis of cell cycle dependent RAI2 protein expression we executed Western blot analysis after cell synchronization and treatment with genotoxic agents. Localization of ectopically expressed HA-tagged RAI2 protein was analyzed in breast cancer cells treated with genotoxic agents using immune fluorescence staining. Finally, we performed in silico validation in different breast cancer data sets. Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability, associated with loss of acentric chromosomal fragments during ana- and metaphase as well as a prolonged mitosis. Furthermore, karyotyping revealed a significant reduction of chromosome number from 78 to 73 in RAI2 depleted cells. In synchronized parental breast cancer cells we could show an increased RAI2 expression during S/G2 phase compared to unsynchronized cells. Interestingly, genotoxic treatment resulted in a significant upregulation of RAI2 protein, which can also be seen as an increased formation of nuclear speckles (p<0.05). Finally, in silico analysis of breast cancer data sets revealed a significant correlation between low RAI2 expression and an expression signature of chromosomal instability (p>0,001). Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability. Our results show that RAI2 depletion causes pre-mitotic DNA lesions. Moreover, in breast cancer patients a low RAI2 expression was significantly associated with chromosomal instability. We conclude that the RAI2 protein preserves mitotic fidelity and chromosomal integrity in luminal breast cancer cell lines by affecting either the DNA damage response or DNA replication during S- and/or G2-phase. Citation Format: Lena Boettcher, Bernd Zobiak, Kerstin Borgmann, Alexandra Zielinski, Antonio Virgilio Failla, Klaus Pantel, Harriet Wikman, Stefan Werner. RAI2 maintains genome stability of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3364.