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Chromosome Research : An International... Aug 2023Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian... (Review)
Review
Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian cancer (HGSOC), one of the most chromosomally unstable tumour types, has a 5-year survival rate of only ~30% - largely due to late diagnosis and rapid development of drug resistance, e.g., via CIN-driven ABCB1 translocations. However, CIN is also a cell cycle vulnerability that can be exploited to specifically target tumour cells, illustrated by the success of PARP inhibitors to target homologous recombination deficiency (HRD). However, a lack of appropriate models with ongoing CIN has been a barrier to fully exploiting disease-specific CIN mechanisms. This barrier is now being overcome with the development of patient-derived cell cultures and organoids. In this review, we describe our progress building a Living Biobank of over 120 patient-derived ovarian cancer models (OCMs), predominantly from HGSOC. OCMs are highly purified tumour fractions with extensive proliferative potential that can be analysed at early passage. OCMs have diverse karyotypes, display intra- and inter-patient heterogeneity and mitotic abnormality rates far higher than established cell lines. OCMs encompass a broad-spectrum of HGSOC hallmarks, including a range of p53 alterations and BRCA1/2 mutations, and display drug resistance mechanisms seen in the clinic, e.g., ABCB1 translocations and BRCA2 reversion. OCMs are amenable to functional analysis, drug-sensitivity profiling, and multi-omics, including single-cell next-generation sequencing, and thus represent a platform for delineating HGSOC-specific CIN mechanisms. In turn, our vision is that this understanding will inform the design of new therapeutic strategies.
Topics: Humans; Female; BRCA1 Protein; Biological Specimen Banks; BRCA2 Protein; Chromosome Disorders; Ovarian Neoplasms; Translocation, Genetic; Chromosomal Instability
PubMed: 37592171
DOI: 10.1007/s10577-023-09731-x -
Journal of Ovarian Research Aug 2023Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance mice detected meiotic arrest of...
Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription-replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.
Topics: Animals; Mice; Cell Cycle; Cell Division; DNA Repair; Fanconi Anemia; Germ Cells; Mice, Knockout; Fanconi Anemia Complementation Group E Protein
PubMed: 37563658
DOI: 10.1186/s13048-023-01252-9 -
Molecular Therapy. Nucleic Acids Sep 2023Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and...
Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A () promoter. When tested in mice, our promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.
PubMed: 37547292
DOI: 10.1016/j.omtn.2023.07.016 -
Frontiers in Plant Science 2023A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic...
A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic memory that can be transmitted both meiotically and mitotically. Such epigenetic memories may increase phenotypic variation to global warming and provide time for adaptation to occur through classical genetic selection. The goal of this study was to understand how warmer temperature conditions experienced during sexual and asexual reproduction affect the transcriptomes of different strawberry () ecotypes. We let four European ecotypes reproduce at two contrasting temperatures (18 and 28°C), either asexually through stolon formation for several generations, or sexually by seeds (achenes). We then analyzed the transcriptome of unfolding leaves, with emphasis on differential expression of genes belonging to the epigenetic machinery. For asexually reproduced plants we found a general transcriptomic response to temperature conditions but for sexually reproduced plants we found less significant responses. We predicted several splicing isoforms for important genes (e.g. a SOC1, LHY, and SVP homolog), and found significantly more differentially presented splicing event variants following asexual vs. sexual reproduction. This difference could be due to the stochastic character of recombination during meiosis or to differential creation or erasure of epigenetic marks during embryogenesis and seed development. Strikingly, very few differentially expressed genes were shared between ecotypes, perhaps because ecotypes differ greatly both genetically and epigenetically. Genes related to the epigenetic machinery were predominantly upregulated at 28°C during asexual reproduction but downregulated after sexual reproduction, indicating that temperature-induced change affects the epigenetic machinery differently during the two types of reproduction.
PubMed: 37521931
DOI: 10.3389/fpls.2023.1213311 -
The American Journal of Dermatopathology Sep 2023GLI1 gene alterations (rearrangement or amplification) have been found in several bone and soft tissue tumors including pericytic tumors, gastric plexiform fibromyxoma,...
GLI1 gene alterations (rearrangement or amplification) have been found in several bone and soft tissue tumors including pericytic tumors, gastric plexiform fibromyxoma, gastroblastoma, and a various group of epithelioid tumors with regional recurrence or distant metastasis. In this article, we describe a case of primary cutaneous epithelioid mesenchymal tumor harboring hitherto not reported ATP2B4::GLI1 gene fusion. A 42-year-old man presented with a growing firm lesion on the left postauricular scalp. Microscopically, the shave biopsy specimen revealed a dermal-based nodular proliferation of relatively monotonous epithelioid cells with round to ovoid nuclei and pale eosinophilic cytoplasm, accompanied by prominent stromal vasculature. Significant cytologic atypia, necrosis, and mitotic activity were absent. The tumor cells were partially positive for CD34 and S-100 protein, but were negative for other markers, including SOX-10, keratins, and myogenic markers. An ATP2B4::GLI1 gene fusion was identified by next-generation sequencing. Array CGH was also performed, but it did not show relevant chromosomal copy number changes. Awareness of this rare cutaneous tumor, and thus, reporting of additional cases is necessary for further delineating its full clinicopathologic spectrum.
Topics: Male; Humans; Adult; Zinc Finger Protein GLI1; Skin Neoplasms; Soft Tissue Neoplasms; Gene Fusion; S100 Proteins; Biomarkers, Tumor; Plasma Membrane Calcium-Transporting ATPases
PubMed: 37506273
DOI: 10.1097/DAD.0000000000002497 -
Proceedings of the National Academy of... Aug 2023PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes...
PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, ) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 mice are chimeras with mixed (neoR retention) cells that act similarly to . Pure F2 embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to cells, genotype and expression-validated pure cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of cells to base damages and Topo inhibitors exceed controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.
Topics: Female; Pregnancy; Animals; Mice; Causality; Alleles; Genotype; Genomic Instability; ADP-Ribosylation
PubMed: 37487079
DOI: 10.1073/pnas.2301972120 -
Histopathology Nov 2023NUTM1-rearranged sarcoma is an emerging entity that differs from NUT carcinoma at the molecular level, with most of the former tumours harbouring fusions involving genes... (Review)
Review
AIMS
NUTM1-rearranged sarcoma is an emerging entity that differs from NUT carcinoma at the molecular level, with most of the former tumours harbouring fusions involving genes in the MYC-associated factor X dimerization (MAD) transcription family (MXD1, MXD4, MXI1 [or MXD2], and MGA). MGA::NUTM1 is one of the most recently described novel gene fusions associated with NUTM1-rearranged sarcoma. Herein we describe the clinicopathologic features of three sarcomas with an MGA::NUTM1 fusion.
METHODS AND RESULTS
The three study patients were male, with an age range of 10-28 years. The tumour sites were deep soft tissue of the thigh, the chest wall, and the pelvis. All three tumours were aggressive, with multiple recurrences and metastases. Histologically, the tumours were composed of monotonous spindle, round, or epithelioid cells in variably hyalinized stroma and prominent aggregates of amianthoid fibre-like collagen or collagen rosettes. Mitotic activity was relatively low (5-12 mitotic figures per 10 hhpf). All tumours tested expressed NUT, with one tumour having S100 protein expression and two tumours having CD99 and CD56 expression. The genetic breakpoints were MGA exon 21, MGA exon 22, and NUTM1 exon 3.
CONCLUSION
MGA::NUTM1 sarcoma often exhibits hyalinized stroma with amianthoid fibre-like collagen or collagen rosettes in the presence of monotonous round, epithelioid, or spindle cell morphology. NUT immunohistochemistry and molecular testing can help confirm the diagnosis.
Topics: Humans; Male; Child; Adolescent; Young Adult; Adult; Female; Neoplasm Proteins; Transcription Factors; Sarcoma; Gene Fusion; Soft Tissue Neoplasms; Collagen; Oncogene Proteins, Fusion; Repressor Proteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
PubMed: 37442637
DOI: 10.1111/his.15004 -
Journal of Medical Virology Jul 2023Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the...
Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.
Topics: Mice; Animals; Humans; Human papillomavirus 16; CRISPR-Cas Systems; Antimicrobial Peptides; Mice, Inbred C57BL; Oncogenes
PubMed: 37403986
DOI: 10.1002/jmv.28934 -
Experimental Cell Research Sep 2023Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated...
Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated internally by chemicals and enzymes, in the absence of exogenous agents, though the sources and consequences of such endogenously generated DSBs remain poorly understood. In the current study, we have investigated the impact of reduced recombinational repair of endogenous DSBs on stress responses, cell morphology and other physical properties of S. cerevisiae (budding yeast) cells. Use of phase contrast and DAPI-based fluorescence microscopy combined with FACS analysis confirmed that recombination-deficient rad52 cell cultures exhibit chronically high levels of G phase cells. Cell cycle phase transit times during G, S and M were similar in WT and rad52 cells, but the length of G phase was increased by three-fold in the mutants. rad52 cells were larger than WT in all phases of the cycle and displayed other quantifiable changes in physical characteristics. The high G cell phenotype was abolished when DNA damage checkpoint genes, but not spindle assembly checkpoint genes, were co-inactivated with RAD52. Several other RAD52 group mutants (rad51, rad54, rad55, rad57 and rad59) also exhibited the high G cell phenotype. The results indicate that recombination deficiency leads to accumulation of unrepaired DSBs during normal mitotic growth that activate a major stress response and produce distinct changes in cellular physiology and morphology.
Topics: Saccharomyces cerevisiae; DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Rad51 Recombinase; DNA Repair; Cell Cycle; Homologous Recombination
PubMed: 37393982
DOI: 10.1016/j.yexcr.2023.113701 -
The International Journal of... Aug 2023Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous...
Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous recombination (HR) deficiency, based on synthetic lethality concept. However, ∼90% of breast cancers are BRCA-wild type; they repair PARPi mediated damage through HR, leading to intrinsic de novo resistance. Hence, there is an unmet need of exploring novel targets in HR-proficient aggressive breast cancers for PARPi treatment. RECQL5 physically interacts and disrupts RAD51 from pre-synaptic filaments, aiding HR resolution, replication fork protection and preventing illegitimate recombination. In the current investigation, we show that targeted inhibition of HR by stabilization of RAD51-RECQL5 complex by a pharmacological inhibitor of RECQL5 (4a; 1,3,4-oxadiazole derivative) in the presence of PARPi [talazoparib (BMN673)] leads to abolition of functional HR with uncontrolled activation of NHEJ repair. This was assessed by GFP based NHEJ reporter assay, KU80 recruitment and in vitro NHEJ based plasmid ligation assay. Concomitant treatment with talazoparib and 4a generates copious amounts of replication stress, prolonged cell cycle arrest, extensive double strand breaks (DSBs) and mitotic catastrophe, leading to sensitization of HR-proficient breast cancers. Suppression of NHEJ activity abolishes 4a-mediated sensitization of breast cancers to PARPi treatment. Imperatively, 4a was ineffective against normal mammary epithelial cells, which expresses low RECQL5 vis-à-vis breast cancer cells. Moreover, functional inhibition of RECQL5 suppresses metastatic potential of breast cancer cells in response to PARPi. Together, we identified RECQL5 as a novel pharmacological target for expanding PARPi based treatment horizon for HR-proficient cancers.
Topics: Humans; Female; Poly(ADP-ribose) Polymerase Inhibitors; Breast Neoplasms; DNA End-Joining Repair; Breast; DNA Replication; Cell Line, Tumor; Homologous Recombination; RecQ Helicases
PubMed: 37392863
DOI: 10.1016/j.biocel.2023.106443