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Science (New York, N.Y.) Aug 2023Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas...
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9-based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes and and and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.
Topics: Humans; Cell Cycle Proteins; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Exonucleases; HEK293 Cells; Mitosis; Poly(ADP-ribose) Polymerases
PubMed: 37440612
DOI: 10.1126/science.adh3694 -
Proceedings of the National Academy of... Jun 2023Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how...
Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating FF-ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal.
Topics: Animals; Humans; Reactive Oxygen Species; Zebrafish; Mitochondria; Epithelial Cells; Adenosine Triphosphate
PubMed: 37276417
DOI: 10.1073/pnas.2216310120 -
Nature Jul 2023In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle, when mitogen signalling engages a positive...
In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.
Topics: Animals; Cell Cycle; Cyclin A2; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; G2 Phase; Mitogens; Mitosis; Phosphorylation; Retinoblastoma Protein; Cyclin-Dependent Kinase 6; S Phase; G1 Phase
PubMed: 37407814
DOI: 10.1038/s41586-023-06274-3 -
Cell Reports Jul 2023The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct...
The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11- and EXO1-mediated nascent DNA degradation from PrimPol-generated, single-stranded DNA (ssDNA) gaps. ATRi also exposes template ssDNA through fork uncoupling and nascent DNA degradation. Electron microscopy reveals that ATRi reduces reversed forks by increasing gap-dependent nascent DNA degradation. At new forks, ATRi triggers MRE11- and CtIP-initiated template DNA degradation by EXO1, exposing nascent ssDNA. Upon PARP inhibition, ATRi preferentially exacerbates gap-dependent nascent DNA degradation at ongoing forks in BRCA1/2-deficient cells and disrupts the restored gap protection in BRCA1-deficient, PARP-inhibitor-resistant cells. Thus, ATR protects ongoing and new forks through distinct mechanisms, providing an extended view of ATR's functions in stabilizing replication forks.
Topics: BRCA1 Protein; BRCA2 Protein; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Humans; Ataxia Telangiectasia Mutated Proteins
PubMed: 37454295
DOI: 10.1016/j.celrep.2023.112792 -
Nature Apr 2024An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination...
An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.
Topics: Humans; DNA Breaks, Double-Stranded; DNA Replication; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Recombinational DNA Repair; S Phase; Transcription, Genetic; Neoplasms; Poly (ADP-Ribose) Polymerase-1
PubMed: 38509368
DOI: 10.1038/s41586-024-07217-2 -
Journal of Alzheimer's Disease Reports 2023Immunotherapeutic efforts to slow the clinical progression of Alzheimer's disease (AD) by lowering brain amyloid-β (Aβ) have included Aβ vaccination, intravenous... (Review)
Review
Immunotherapeutic efforts to slow the clinical progression of Alzheimer's disease (AD) by lowering brain amyloid-β (Aβ) have included Aβ vaccination, intravenous immunoglobulin (IVIG) products, and anti-Aβ monoclonal antibodies. Neither Aβ vaccination nor IVIG slowed disease progression. Despite conflicting phase III results, the monoclonal antibody Aducanumab received Food and Drug Administration (FDA) approval for treatment of AD in June 2021. The only treatments unequivocally demonstrated to slow AD progression to date are the monoclonal antibodies Lecanemab and Donanemab. Lecanemab received FDA approval in January 2023 based on phase II results showing lowering of PET-detectable Aβ; phase III results released at that time indicated slowing of disease progression. Topline results released in May 2023 for Donanemab's phase III trial revealed that primary and secondary end points had been met. Antibody binding to Aβ facilitates its clearance from the brain via multiple mechanisms including promoting its microglial phagocytosis, activating complement, dissolving fibrillar Aβ, and binding of antibody-Aβ complexes to blood-brain barrier receptors. Antibody binding to Aβ in peripheral blood may also promote cerebral efflux of Aβ by a peripheral sink mechanism. According to the amyloid hypothesis, for Aβ targeting to slow AD progression, it must decrease downstream neuropathological processes including tau aggregation and phosphorylation and (possibly) inflammation and oxidative stress. This review discusses antibody-mediated mechanisms of Aβ clearance, findings in AD trials involving Aβ vaccination, IVIG, and anti-Aβ monoclonal antibodies, downstream effects reported in those trials, and approaches which might improve the Aβ-clearing ability of monoclonal antibodies.
PubMed: 37662616
DOI: 10.3233/ADR-230025 -
Molecular Cell Feb 2024Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and...
Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.
Topics: Humans; BRCA1 Protein; BRCA2 Protein; DNA Damage; DNA Repair; DNA Repair Enzymes; Exodeoxyribonucleases; Homologous Recombination; Neoplasms
PubMed: 38266640
DOI: 10.1016/j.molcel.2023.12.039 -
Cancer Research Sep 2023Estrogen-related receptor β (ESRRB) is a member of the orphan nuclear receptor family and mediates stem cell self-renewal and early embryonic development. Previous...
UNLABELLED
Estrogen-related receptor β (ESRRB) is a member of the orphan nuclear receptor family and mediates stem cell self-renewal and early embryonic development. Previous studies have also reported that ESRRB plays a role in the development and progression of breast cancer and prostate cancer. In this study, we observed that ESRRB was highly expressed in cervical cancer and was associated with disease progression. Knocking out ESRRB using CRISPR/Cas9 gene editing in cervical cancer cells induced cell-cycle arrest at the transition from the G0-G1 phase to the S phase, resulting in inhibition of cell proliferation in vitro and reduced tumor growth in vivo. Conversely, ectopic expression of ESRRB significantly promoted the proliferation of cervical cancer cells. ESRRB activated transcription of SMAD7, a TGFβ pathway inhibitor, which blocked phosphorylation and nuclear translocation of SMAD2/3 to the nucleus, thereby downregulating CDKN1A and upregulating CCNA2 and MYC. In turn, MYC transactivated ESRRB and upregulated SMAD7, thus forming a positive feedback loop with ESRRB. Together, these findings identify the tumor-promoting function of ESRRB in cervical cancer and reveal a mechanism by which ESRRB stimulates cell proliferation to promote cancer progression.
SIGNIFICANCE
The ESRRB/SMAD7/MYC-positive feedback loop inhibits TGFβ signaling to activate cell-cycle progression and promote proliferation in cervical cancer, thereby driving tumor growth.
Topics: Female; Humans; Cell Proliferation; Receptors, Estrogen; Signal Transduction; Transforming Growth Factor beta; Uterine Cervical Neoplasms
PubMed: 37350664
DOI: 10.1158/0008-5472.CAN-23-0067 -
Nature Nov 2023In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements...
In eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 trimethylation (H3K9me3). Loss of silencing of the repeat elements leads to genome instability and human diseases, including cancer and ageing. Although the role of H3K9me3 in the establishment and maintenance of heterochromatin silencing has been extensively studied, the pattern and mechanism that underlie the partitioning of parental H3K9me3 at replicating DNA strands are unknown. Here we report that H3K9me3 is preferentially transferred onto the leading strands of replication forks, which occurs predominantly at long interspersed nuclear element (LINE) retrotransposons (also known as LINE-1s or L1s) that are theoretically transcribed in the head-on direction with replication fork movement. Mechanistically, the human silencing hub (HUSH) complex interacts with the leading-strand DNA polymerase Pol ε and contributes to the asymmetric segregation of H3K9me3. Cells deficient in Pol ε subunits (POLE3 and POLE4) or the HUSH complex (MPP8 and TASOR) show compromised H3K9me3 asymmetry and increased LINE expression. Similar results were obtained in cells expressing a MPP8 mutant defective in H3K9me3 binding and in TASOR mutants with reduced interactions with Pol ε. These results reveal an unexpected mechanism whereby the HUSH complex functions with Pol ε to promote asymmetric H3K9me3 distribution at head-on LINEs to suppress their expression in S phase.
Topics: Humans; DNA Replication; Gene Silencing; Histones; Long Interspersed Nucleotide Elements; Lysine; Methylation; S Phase
PubMed: 37938774
DOI: 10.1038/s41586-023-06711-3 -
Signal Transduction and Targeted Therapy Jul 2023Due to the essential role of cyclin D1 in regulating transition from G1 to S phase in cell cycle, aberrant cyclin D1 expression is a major oncogenic event in many types...
Due to the essential role of cyclin D1 in regulating transition from G1 to S phase in cell cycle, aberrant cyclin D1 expression is a major oncogenic event in many types of cancers. In particular, the dysregulation of ubiquitination-dependent degradation of cyclin D1 contributes to not only the pathogenesis of malignancies but also the refractory to cancer treatment regiments with CDK4/6 inhibitors. Here we show that in colorectal and gastric cancer patients, MG53 is downregulated in more than 80% of tumors compared to the normal gastrointestinal tissues from the same patient, and the reduced MG53 expression is correlated with increased cyclin D1 abundance and inferior survival. Mechanistically, MG53 catalyzes the K48-linked ubiquitination and subsequent degradation of cyclin D1. Thus, increased expression of MG53 leads to cell cycle arrest at G1, and thereby markedly suppresses cancer cell proliferation in vitro as well as tumor growth in mice with xenograft tumors or AOM/DSS induced-colorectal cancer. Consistently, MG53 deficiency results in accumulation of cyclin D1 protein and accelerates cancer cell growth both in culture and in animal models. These findings define MG53 as a tumor suppressor via facilitating cyclin D1 degradation, highlighting the therapeutic potential of targeting MG53 in treating cancers with dysregulated cyclin D1 turnover.
Topics: Humans; Animals; Mice; Ubiquitin-Protein Ligases; Cyclin D1; Cell Proliferation; Cell Cycle Checkpoints; Stomach Neoplasms; Membrane Proteins
PubMed: 37414783
DOI: 10.1038/s41392-023-01458-9