RESUMO
Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/CCdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD.
Assuntos
Ciclina E , Duplicação Gênica , Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Linhagem Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Mitose , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.
Assuntos
Anemia de Fanconi , Neoplasias , Humanos , Sobrevivência Celular/genética , Cromatina/genética , Ciclina E/genética , Ciclina E/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Neoplasias/genéticaRESUMO
The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Replicação do DNA , Proteínas Intrinsicamente Desordenadas/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Fase G1 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Complexo de Reconhecimento de Origem/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estabilidade Proteica , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.
Assuntos
Ciclina E , Proteínas de Membrana , Neoplasias Ovarianas , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Proteína Quinase CDC2 , Ciclina E/genética , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Mutações Sintéticas LetaisRESUMO
E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.
Assuntos
Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Regulação Alostérica , Biocatálise , Microscopia Crioeletrônica , Ciclina E/metabolismo , Humanos , Fosforilação , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.
Assuntos
Instabilidade Genômica , Neoplasias/genética , Nucleotídeos/biossíntese , Ciclina E/metabolismo , Replicação do DNA , Fatores de Transcrição E2F/metabolismo , Humanos , Perda de Heterozigosidade , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotídeos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase SRESUMO
Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.
Assuntos
Instabilidade Cromossômica/genética , Evolução Molecular , Cariótipo , Metástase Neoplásica/genética , Neoplasias/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 8/genética , Células Clonais/metabolismo , Células Clonais/patologia , Ciclina E/genética , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Perda de Heterozigosidade/genética , Masculino , Mutagênese , Metástase Neoplásica/patologia , Neoplasias/patologia , Proteínas Oncogênicas/genéticaRESUMO
Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Neoplasias da Mama , Ciclina E , Replicação do DNA , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Ciclina E/genética , Ciclina E/metabolismo , Replicação do DNA/genética , Polimorfismo de Nucleotídeo Único , Proliferação de Células , Variações do Número de Cópias de DNA , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Idoso , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , AdultoRESUMO
In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. Here we show that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.
Assuntos
Proteínas de Drosophila , Células-Tronco Neurais , Animais , Ciclina E/genética , Ciclina E/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/genéticaRESUMO
Circular RNAs (circRNAs) play important roles in cancer occurrence and progression. To explore and elucidate the clinical significance of specific circular RNA in melanoma and its potential molecular mechanism. CircROR1 expression in melanoma cells and tissues was confirmed by qRT-PCR and ISH. qRT-PCR and Western blotting were performed to measure the levels of CCNE1, KAT2A, MMP9 and TIMP2. MTT, Transwell and wound healing assays were performed to evaluate cell proliferation, invasion and metastasis. A xenograft mouse model was established to further verify the CircROR1/CCNE1 axis in vivo. RNA pull-down and RIP assays were performed to detect the direct interaction KAT2A and CircROR1. A ChIP assay was used to investigate the enrichment of H3K9ac acetylation in the CCNE1 promoter. CircROR1 was significantly upregulated in metastatic melanoma cells and tissues, promoting proliferation, invasion and metastasis in vitro and tumour growth in vivo. CircROR1 overexpression increased CCNE1 and MMP9 protein expression and decreased TIMP2 protein expression. Functional rescue assays demonstrated that CircROR1 played a role in promoting malignant progression through CCNE1. CircROR1 specifically bound to the KAT2A protein without affecting its expression. CircROR1 overexpression increased the level of H3K9ac modification in the CCNE1 promoter region by recruiting KAT2A, thus upregulating CCNE1 expression. CircROR1 upregulates CCNE1 expression through KAT2A-mediated histone acetylation. Our research confirms the critical role of CircROR1 in melanoma invasion and metastasis, and CircROR1 could serve as a potential therapeutic target for melanoma treatment.
Assuntos
Melanoma , MicroRNAs , Humanos , Animais , Camundongos , MicroRNAs/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/metabolismo , Linhagem Celular Tumoral , RNA Circular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ciclina E/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismoRESUMO
Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation.
Assuntos
Ciclina A/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Ciclina A/genética , Ciclina E/genética , Ciclina E/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.
Assuntos
Replicação do DNA , Fase G1/genética , Instabilidade Genômica/genética , Neoplasias/genética , Oncogenes/genética , Origem de Replicação/genética , Fase S/genética , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Estudos de Coortes , Ciclina E/genética , Ciclina E/metabolismo , DNA/biossíntese , DNA/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Humanos , Proteínas Oncogênicas/genética , Transcrição Gênica/genética , Translocação Genética/genéticaRESUMO
Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle1-4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/CCDH1, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/CCDH1, which suggests that APC/CCDH1 inactivation is the point of no return for cell-cycle entry 3 . Rapid inactivation of APC/CCDH1 requires early mitotic inhibitor 1 (EMI1)3,5, but the molecular mechanism that controls this cell-cycle commitment step is unknown. Here we show using human cell models that cell-cycle commitment is mediated by an EMI1-APC/CCDH1 dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/CCDH1. The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/CCDH1 activity during G1 and a state with high EMI1 levels and low APC/CCDH1 activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Our study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/CCDH1 inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/CCDH1 to being an inhibitor of APC/CCDH1.
Assuntos
Proteínas Cdh1/antagonistas & inibidores , Proteínas Cdh1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas F-Box/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas F-Box/genética , Retroalimentação Fisiológica , Fase G1 , Células HeLa , Humanos , Fase SRESUMO
Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Ciclina E/genética , Quebras de DNA de Cadeia Dupla , DNA/genética , Osteossarcoma/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação , Proteína da Polipose Adenomatosa do Colo/deficiência , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina E/metabolismo , DNA/metabolismo , Fase G1 , Expressão Gênica , Instabilidade Genômica , Humanos , Camundongos , Camundongos Knockout , Nocodazol/farmacologia , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Osteossarcoma/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fase S , Estresse Fisiológico , Análise de SobrevidaRESUMO
CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.
Assuntos
Biomarcadores Tumorais , Ciclina E , Rearranjo Gênico , Proteínas Oncogênicas , Proteínas Repressoras , Humanos , Masculino , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/genética , Feminino , Adulto , Ciclina E/metabolismo , Ciclina E/genética , Pessoa de Meia-Idade , Adolescente , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Adulto Jovem , Criança , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Imuno-Histoquímica/métodos , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Sarcoma de Ewing/genética , Sarcoma/patologia , Sarcoma/metabolismo , Sarcoma/genética , Sarcoma/diagnóstico , Hibridização in Situ Fluorescente/métodos , Idoso , Pré-Escolar , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Células Pequenas/metabolismo , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/patologia , Sarcoma de Células Pequenas/diagnósticoRESUMO
Most mesenchymal tumors found in the uterine corpus are benign tumors; however, uterine leiomyosarcoma is a malignant tumor with unknown risk factors that repeatedly recurs and metastasizes. In some cases, the histopathologic findings of uterine leiomyoma and uterine leiomyosarcoma are similar and surgical pathological diagnosis using excised tissue samples is difficult. It is necessary to analyze the risk factors for human uterine leiomyosarcoma and establish diagnostic biomarkers and treatments. Female mice deficient in the proteasome subunit low molecular mass peptide 2 (LMP2)/ß1i develop uterine leiomyosarcoma spontaneously. MATERIAL AND METHODS: Out of 334 patients with suspected uterine mesenchymal tumors, patients diagnosed with smooth muscle tumors of the uterus were selected from the pathological file. To investigate the expression status of biomarker candidate factors, immunohistochemical staining was performed with antibodies of biomarker candidate factors on thin-cut slides of human uterine leiomyosarcoma, uterine leiomyoma, and other uterine mesenchymal tumors. RESULTS AND DISCUSSION: In human uterine leiomyosarcoma, there was a loss of LMP2/ß1i expression and enhanced cyclin E1 and Ki-67/MIB1 expression. In human uterine leiomyomas and normal uterine smooth muscle layers, enhanced LMP2/ß1i expression and the disappearance of the expression of E1 and Ki-67/MIB1 were noted. The pattern of expression of each factor in other uterine mesenchymal tumors was different from that of uterine leiomyosarcoma. CONCLUSIONS: LMP2/ß1i, cyclin E1, and Ki-67/MIB1 may be candidate factors for biomarkers of human uterine leiomyosarcoma. Further large-cohort clinical trials should be conducted to establish treatments and diagnostics for uterine mesenchymal tumors.
Assuntos
Biomarcadores Tumorais , Ciclina E , Leiomioma , Leiomiossarcoma , Proteínas Oncogênicas , Neoplasias Uterinas , Humanos , Feminino , Neoplasias Uterinas/patologia , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Leiomiossarcoma/patologia , Leiomiossarcoma/diagnóstico , Leiomioma/metabolismo , Leiomioma/patologia , Leiomioma/diagnóstico , Leiomioma/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ciclina E/metabolismo , Ciclina E/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Adulto , Cisteína EndopeptidasesRESUMO
Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Epigênese GenéticaRESUMO
BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.
Assuntos
Carcinoma , Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Feminino , Humanos , Neoplasias Ovarianas/patologia , Fatores de Transcrição/genética , RNA Mensageiro , Cistadenocarcinoma Seroso/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/uso terapêutico , Ciclina E/genéticaRESUMO
Cell cycle transitions are controlled by multiple cell cycle regulators, especially CDKs. Several CDKs, including CDK1-4 and CDK6, promote cell cycle progression directly. Among them, CDK3 is critically important because it triggers the transitions of G0 to G1 and G1 to S phase through binding to cyclin C and cyclin E1, respectively. In contrast to its highly related homologs, the molecular basis of CDK3 activation remains elusive due to the lack of structural information of CDK3, particularly in cyclin bound form. Here we report the crystal structure of CDK3 in complex with cyclin E1 at 2.25 Å resolution. CDK3 resembles CDK2 in that both adopt a similar fold and bind cyclin E1 in a similar way. The structural discrepancy between CDK3 and CDK2 may reflect their substrate specificity. Profiling a panel of CDK inhibitors reveals that dinaciclib inhibits CDK3-cyclin E1 potently and specifically. The structure of CDK3-cyclin E1 bound to dinaciclib reveals the inhibitory mechanism. The structural and biochemical results uncover the mechanism of CDK3 activation by cyclin E1 and lays a foundation for structural-based drug design.
Assuntos
Indolizinas , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Quinase 2 Dependente de Ciclina , Indolizinas/farmacologia , Compostos de Piridínio/farmacologia , Ciclo Celular/fisiologia , Ciclina E/metabolismo , Ciclinas/metabolismoRESUMO
Systemic and stem cell niche-emanating cytokines and growth factors can promote regeneration, through mitosis. High mitosis, however, predisposes for all types of cancer and, thus, a trade-off exists between regeneration capacity and tissue homeostasis. Here, we study the role of tissue-intrinsic regenerative signaling in stem cell mitosis of adult Drosophila midgut of different genetic backgrounds. We provide evidence of two naturally occurring types of balance between mitosis and enterocyte nucleus growth: one based mostly on stem cell mitosis producing new cells and the other based mostly on the degree of young enterocyte nucleus size increase. Mitosis promotes intestinal host defense to infection, but predisposes for dysplasia in the form of stem cell-like clusters. Enterocyte nucleus growth also promotes host defense, without the drawback of promoting dysplasia. Through quantitative genetics, we identified eiger as an autocrine and paracrine inducer of stem cell mitosis. eiger expression in immature epithelial cells tilts the balance towards mitosis and dysplasia via a positive-feedback loop of highly mitotic stem cells sustaining more small nucleus enterocytes, which in turn supply more Eiger.