cyclin-d1 and Chromosomal-Instability

Cyclins encode regulatory subunits of holoenzymes that phosphorylate a variety of cellular substrates. Although the classic role of cyclins in cell cycle progression and tumorigenesis has been well characterized, new functions have been identified, including the induction of cellular migration and invasion, enhancement of angiogenesis, inhibition of mitochondrial metabolism, regulation of transcription factor signaling via a DNA-bound form, the induction of chromosomal instability, enhancement of DNA damage sensing and DNA damage repair, and feedback governing expression of the noncoding genome. This review describes the mechanisms of these new functions of cyclin D1.

Topics: Animals; Cell Cycle; Chromosomal Instability; Cyclin D1; DNA Repair; Gene Expression Regulation; Humans; Mice; Mitochondria

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

Topics: Adenocarcinoma; Amino Acid Substitution; Aneuploidy; Animals; Catalytic Domain; Cell Transformation, Neoplastic; Cells, Cultured; Centrosome; Chromosomal Instability; Cyclin D1; Female; Fibroblasts; Genes, bcl-1; Humans; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Knockout; Mice, Transgenic; Mutation; Piperazines; Pyridines; Recombinant Fusion Proteins; Spindle Apparatus; Transduction, Genetic

The histopathology of premalignant laryngeal lesions does not provide reliable information on the risk of malignant transformation, hence we examined new molecular markers which can easily be implemented in clinical practice. Dual-target fluorescence in situ hybridisation (FISH) for chromosome 1 and 7 centromeres was performed on tissue sections of laryngeal premalignancies in 69 patients. Chromosome instability was indicated by numerical imbalances and/or polysomy for chromosomes 1 and 7. Additionally, immunostainings for p53, Cyclin D1 and (p)FADD expression were evaluated. Malignant progression was recorded. Eighteen patients with carcinoma in situ (CIS) were treated after diagnosis and excluded from follow-up. Chromosome instability was strongly associated with a high risk of malignant transformation, especially in lower grade lesions (hyperplasia, mild and moderate dysplasia; odds ratio = 8.4, p = 0.004). Patients with lesions containing chromosome instability showed a significantly worse 5-year progression-free survival than those with premalignancies without chromosome instability (p = 0.002). Neither histopathology nor the protein markers predicted progression in univariate analysis, although histopathological diagnosis, p53 and FADD contributed positively to chromosome instability in multivariate analysis. Chromosome instability is associated with malignant progression of laryngeal premalignancies, especially in lower grade lesions. These results may contribute to better risk counselling, provided that they can be validated in a larger patient set.

Topics: Adult; Aged; Chromosomal Instability; Cyclin D1; Disease Progression; Disease-Free Survival; Fas-Associated Death Domain Protein; Female; Humans; Hyperplasia; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Laryngeal Neoplasms; Larynx; Male; Middle Aged; Precancerous Conditions; Young Adult

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Topics: Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Chromosomal Instability; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Breaks, Double-Stranded; DNA Repair; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kinetochores; MCF-7 Cells; Nuclear Receptor Subfamily 1, Group F, Member 3; Protein Interaction Maps; Protein-Arginine N-Methyltransferases; Receptors, Estrogen; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Tribbles-related protein 3 (TRB3) has been shown to be a crucial modulator of tumorigenesis. However, the precise role and the functional morphology of TRB3 are not clearly understood. To elucidate these enigmas we established the cell line, M2TRB3, by introducing the human TRB3 gene and protein in Cl66M2 (M2) mouse mammary tumor cells. This cell line stably expressed the TRB3 gene and protein. After 72 h of cell culture, there was a 34% increase in the growth of M2TRB3 cells compared to the control M2 mock cells. The mean volume of the tumors originating from the M2TRB3 cells was significantly increased by 38% when compared to the mean volume of the M2 mock tumors, and the proliferating cell nuclear antigen (PCNA) labeling index in the M2TRB3 tumors was higher when compared to that of the M2 and M2 mock cells. In the tumor tissue samples, the mean diameter of nuclei in the M2TRB3 tumor cells (9.4±0.3 µm) showed a significant increase compared to that of the M2 mock tumor cells (7.0±0.2 µm). M2TRB3 cells also showed a marked increase in the population of tetraploid or octaploid nuclei compared to M2 mock cells bearing mainly either diploid or tetraploid nuclei. Western blot analysis revealed the overexpression of cyclin B1 and cyclin D1 in M2TRB3 cells when compared to that in the M2 mock cells. These novel findings provide further evidence that TRB3 promotes cell proliferation and chromosomal instability by causing polyploidization during development.

Topics: Animals; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line; Cell Proliferation; Chromosomal Instability; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mammary Neoplasms, Animal; Mice; RNA, Messenger

The Pin1 prolyl isomerase regulates phosphorylation signaling by controlling protein conformation after phosphorylation, and its upregulation promotes oncogenesis via acting on numerous oncogenic molecules. SUMOylation and deSUMOylation are dynamic mechanisms regulating a spectrum of protein activities. The SUMO proteases (SENP) remove SUMO conjugate from proteins, and their expression is deregulated in cancers. However, nothing is known about the role of SUMOylation in regulating Pin1 function. Here, we show that Pin1 is SUMOylated on Lys6 in the WW domain and on Lys63 in the PPIase domain. Pin1 SUMOylation inhibits its protein activity and oncogenic function. We further identify that SENP1 binds to and deSUMOylates Pin1. Importantly, either overexpression of SENP1 or disruption of Pin1 SUMOylation promotes the ability of Pin1 to induce centrosome amplification and cell transformation. Moreover, SENP1 also increases Pin1 protein stability in cell cultures, and Pin1 levels are positively correlated with SENP1 levels in human breast cancer specimens. These results not only uncover Pin1 SUMOylation on Lys6/63 as a novel mechanism to inhibit its activity and function but also identify a critical role for SENP1-mediated deSUMOylation in promoting Pin1 function during tumorigenesis.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Centrosome; Chromosomal Instability; Cyclin D1; Cysteine Endopeptidases; Endopeptidases; Female; Gene Knockdown Techniques; Humans; Mice; NIMA-Interacting Peptidylprolyl Isomerase; Oxidative Stress; Peptidylprolyl Isomerase; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Sumoylation

The cell has many mechanisms for protecting the integrity of its genome. These mechanisms are often weakened or absent in many cancers, leading to high rates of chromosomal instability in tumors. Control of the cell cycle is crucial for the function of these checkpoints, and is frequently lost in cancers as well. Overexpression of Cyclin D1 in a large number of breast cancers causes overactivation of the cyclin-dependent kinases, including Cdk2. Constitutive Cdk2 activation through Cyclin D1 generates tumors in mice that are aneuploid and have many characteristics indicative of chromosomal instability. Expression of these complexes in the MCF10A cell line leads to retinoblastoma protein (Rb) hyperphosphorylation, a subsequent increase in proliferation rate, and increased expression of the spindle assembly checkpoint protein Mad2. This results in a strengthening of the spindle assembly checkpoint and renders cells more sensitive to the spindle poison paclitaxel. Constitutive Rb phosphorylation also causes a weakening of the p53-dependent tetraploidy checkpoint. Cells with overactive Cdk2 fail to arrest after mitotic slippage in the presence of paclitaxel or cytokinesis failure during treatment with cytochalasin-B, generating 8N populations. This additional increase in DNA content appears to further intensify the tetraploidy checkpoint in a step-wise manner. These polyploid cells are not viable long-term, either failing to undergo division or creating daughter cells that are unable to undergo subsequent division. This study raises intriguing questions about the treatment of tumors with overactive Cdk2.

Topics: Aneuploidy; Cell Cycle Checkpoints; Cell Line, Tumor; Centrosome; Chromosomal Instability; Cyclin D1; Cyclin-Dependent Kinase 2; Flow Cytometry; Humans; Immunoblotting; Microscopy, Fluorescence; Spindle Apparatus; Tetraploidy

Subgroups of patients with oral pre-malignant lesions (OPLs) are at extremely high risk for developing invasive cancer in spite of surgical excision. The objective of this study was to evaluate the utility of specific genes and their associated centromeres as markers to stratify OPLs for their cancer risk. Samples used in this study included 35 oral dysplasia with known outcome and 20 normal oral mucosa. Of the dysplasias, 20 were from an ongoing longitudinal study showing progression. The remaining 15 cases (2 of which progressed) were chosen from the population-based, provincial BC Oral Biopsy Service (OBS). Copy number alterations at EGFR, CEP7, CCND1, and CEP11 were evaluated by fluorescent in situ hybridization (FISH). There was no significant difference in demographics between progressors and non-progressors. Specific FISH profiles at these genes and their corresponding centromeres were associated with progression. High gene gain of CCND1 was associated with an 8-fold elevated risk of progression compared with those with no gain in time-to-progression analysis. Numerical alterations of EGFR and CCND1 and their centromeres might be an effective means for identifying OPLs at risk. Future studies will expand on this analysis and set the stage for application of this approach in routine clinical practice.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Cell Transformation, Neoplastic; Centromere; Chromosomal Instability; Cyclin D1; Disease Progression; ErbB Receptors; Female; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Polyploidy; Precancerous Conditions; Proportional Hazards Models; Retrospective Studies; Risk Factors; Sensitivity and Specificity

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; Chromosomal Instability; Chromosome Aberrations; Cyclin D1; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Karyotyping; Mice; Mice, Transgenic; Transcription, Genetic

We developed mouse model systems to investigate the potential for cyclin D1 to induce CIN in vivo. In a mammary gland specific Tet-inducible model the acute expression profile regulated by cyclin D1 after 7 days was enriched in genes that rank highly with CIN. We also used a mammary gland targeted model (MMTV) to continuously express cyclin D1. The mice started to develop mammary gland tumors at 400 days and the tumor-free incidence was 40% in MMTV-cyclin D1. The gene expression profile of the tumors showed enrichment for the CIN signature. We next compared cyclin D1 expression and the highest ranking CIN genes to a breast cancer expression database and discovered that expression of genes promoting CIN are highly enriched in luminal subtype and that high cyclin D1 and CIN expression correlate specifically in the luminal B subtype. There is increasing interest in employing drugs in the clinic that exploit CIN in tumors. The high CIN expression index in luminal B breast cancer provides a basis for using Cdk and CIN inhibitors as a targeted therapeutic approach.

Topics: Animals; Chromatin Assembly and Disassembly; Chromosomal Instability; Cyclin D1; Gene Knockout Techniques; Humans; Mice; Protein Binding; Signal Transduction; Up-Regulation

Aneuploidy, centrosome abnormalities and gene amplification are hallmarks of chromosome instability (CIN) in cancer. Yet there are no studies of the in vivo behavior of these phenomena within the same bladder tumor.. Twenty-one paraffin-embedded bladder tumors were analyzed by conventional comparative genome hybridization and fluorescence in situ hybridization (FISH) with a cyclin D1 gene (CCND1)/centromere 11 dual-color probe. Immunofluorescent staining of alpha, beta and gamma tubulin was also performed.. Based on the CIN index, defined as the percentage of cells not displaying the modal number for chromosome 11, tumors were classified as CIN-negative and CIN-positive. Fourteen out of 21 tumors were considered CIN-positive. All T1G3 tumors were included in the CIN-positive group whereas the majority of Ta samples were classified as CIN-negative tumors. Centrosome clustering was observed in six out of 12 CIN-positive tumors analyzed. CCND1 amplification in homogeneously staining regions was present in six out of 14 CIN-positive tumors; three of them also showed amplification of this gene in double minutes.. Complex in vivo behavior of CCND1 amplicon in bladder tumor cells has been demonstrated by accurate FISH analysis on paraffin-embedded tumors. Positive correlation between high heterogeneity, centrosome abnormalities and CCND1 amplification was found in T1G3 bladder carcinomas. This is the first study to provide insights into the coexistence of CCND1 amplification in homogeneously staining regions and double minutes in primary bladder tumors. It is noteworthy that those patients whose tumors showed double minutes had a significantly shorter overall survival rate (p < 0.001).

Topics: Adult; Aged; Biomarkers, Tumor; Centrosome; Chromosomal Instability; Chromosomes, Human, Pair 11; Comparative Genomic Hybridization; Cyclin D1; Female; Fluorescent Antibody Technique; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Male; Micronuclei, Chromosome-Defective; Middle Aged; Mitosis; Neoplasm Staging; Paraffin Embedding; Prognosis; Time Factors; Tubulin; Urinary Bladder Neoplasms

Ovarian surface epithelium (OSE) is considered to give rise to epithelial ovarian carcinomas (EOCs). To elucidate early processes contributing to the development of EOCs from the OSE, two batches of primary human OSE cells were transduced with non-viral human genes (mutant Cdk4, cyclinD1 and hTERT) so as to efficiently establish normal diploid OSE cells without chromosomal instability. Then defined genetic alterations frequently observed in EOCs were transduced into the OSE cells. A combination of p53 inactivation and oncogenic Kras transduction did not confer tumor-forming ability in immunodeficient mice, though additional transduction of Akt or combined transduction of c-myc with bcl-2 did result in tumor formation. In the latter case, tumors demonstrated phenotypes reminiscent of human EOCs, including cytokeratin expression, a highly aggressive phenotype, metastatic behavior and formation of ascites. These results indicate that inactivation of p53 and activation of the Ras pathway play critical roles in ovarian carcinogenesis in co-operation with the Akt or c-myc pathways. This first in vitro model system faithfully recapitulating the development of EOCs using normal human OSE cells should greatly facilitate further studies of EOCs.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomal Instability; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial Cells; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Oncogenes; Ovarian Neoplasms; Ovary; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; ras Proteins; Telomerase; Transduction, Genetic; Tumor Suppressor Protein p53

SNF5 is a core subunit of the SWI/SNF chromatin-remodeling complex. Mammalian SNF5 is essential for normal cell viability, and loss or mutation of the human SNF gene is the molecular basis for familial malignant rhabdoid tumorigenesis. Previous studies have suggested that SNF5 suppresses cancer by signaling through the p16Ink4a and retinoblastoma tumor suppressors to negatively regulate cell cycle progression from G0/G1 into S phase. A recent paper in Genes & Development (Vries et al., 2005) reports that human SNF5 also signals via the p16INK4a-Rb-E2F pathway to regulate chromosomal stability, suggesting a new function for this chromatin remodeling protein in tumor suppression.

Topics: Aneuploidy; Animals; Cell Cycle; Cell Cycle Proteins; Chromatin Assembly and Disassembly; Chromosomal Instability; Chromosomal Proteins, Non-Histone; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; Humans; Models, Biological; Phosphorylation; Polyploidy; Proto-Oncogene Proteins; Retinoblastoma Protein; Rhabdoid Tumor; SMARCB1 Protein; Transcription Factors; Tumor Suppressor Proteins