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Nature Mar 2020Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis). Gasdermin E (GSDME, also known as...
Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis). Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8 T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.
Topics: Animals; Apoptosis; Aspartic Acid; Cell Line, Tumor; Female; Granzymes; Humans; Loss of Function Mutation; Mice; Neoplasms; Pyroptosis; Receptors, Estrogen; T-Lymphocytes, Cytotoxic
PubMed: 32188940
DOI: 10.1038/s41586-020-2071-9 -
Genome Research Feb 2022Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the...
Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at ∼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, = 28) than in plasma of patients with cancer (median = 14.2%, = 21, < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers ( < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.
Topics: Biomarkers, Tumor; Cell-Free Nucleic Acids; DNA; DNA, Single-Stranded; Humans; Neoplasms; Sequence Analysis, DNA
PubMed: 34930798
DOI: 10.1101/gr.275691.121 -
The Journal of Experimental Medicine Jan 2023Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we...
Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent.
Topics: Humans; Angiopoietin-Like Protein 4; Angiopoietins; Biomarkers, Tumor; Neoplasms; Peptide Fragments
PubMed: 36269299
DOI: 10.1084/jem.20202595 -
Annals of the New York Academy of... Nov 2019Chromogranin A (CgA)-the index member of the chromogranin/secretogranin secretory protein family-is ubiquitously distributed in endocrine, neuroendocrine, and immune... (Review)
Review
Chromogranin A (CgA)-the index member of the chromogranin/secretogranin secretory protein family-is ubiquitously distributed in endocrine, neuroendocrine, and immune cells. Elevated levels of CgA-related polypeptides, consisting of full-length molecules and fragments, are detected in the blood of patients suffering from neuroendocrine tumors, heart failure, renal failure, hypertension, rheumatoid arthritis, and inflammatory bowel disease. Full-length CgA and various CgA-derived peptides, including vasostatin-1, pancreastatin, catestatin, and serpinin, are expressed at different relative levels in normal and pathological conditions and exert diverse, and sometime opposite, biological functions. For example, CgA is overexpressed in genetic hypertension, whereas catestatin is diminished. In rodents, the administration of catestatin decreases hypertension, cardiac contractility, obesity, atherosclerosis, and inflammation, and it improves insulin sensitivity. By contrast, pancreastatin is elevated in diabetic patients, and the administration of this peptide to obese mice decreases insulin sensitivity and increases inflammation. CgA and the N-terminal fragment of vasostatin-1 can enhance the endothelial barrier function, exert antiangiogenic effects, and inhibit tumor growth in animal models, whereas CgA fragments lacking the CgA C-terminal region promote angiogenesis and tumor growth. Overall, the CgA system, consisting of full-length CgA and its fragments, is emerging as an important and complex player in cardiovascular, immunometabolic, and cancer regulation.
Topics: Animals; Cardiovascular System; Chromogranin A; Humans; Inflammation; Neoplasms; Peptide Fragments
PubMed: 31588572
DOI: 10.1111/nyas.14249 -
Proceedings of the National Academy of... May 2023During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or...
During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells, respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation and accumulate cytoplasm and organelles from sister germ cells to become primary oocytes. Changes in cyst structure and differential cell volumes among cyst germ cells suggest that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During fetal cyst formation, E-cadherin (E-cad) junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cad-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cad knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on how oocyte fate is determined within mouse germline cysts.
Topics: Male; Female; Animals; Mice; Oocytes; Germ Cells; Cytoplasm; Organelles; Gametogenesis; Cysts; Oogenesis
PubMed: 37155904
DOI: 10.1073/pnas.2219683120 -
Essays in Biochemistry Sep 2023The DNA sensor cGAS (cyclic GMP-AMP synthase) and its adaptor protein STING (Stimulator of Interferon Genes) detect the presence of cytosolic DNA as a sign of infection... (Review)
Review
The DNA sensor cGAS (cyclic GMP-AMP synthase) and its adaptor protein STING (Stimulator of Interferon Genes) detect the presence of cytosolic DNA as a sign of infection or damage. In cancer cells, this pathway can be activated through persistent DNA damage and chromosomal instability, which results in the formation of micronuclei and the exposure of DNA fragments to the cytosol. DNA damage from radio- or chemotherapy can further activate DNA sensing responses, which may occur in the cancer cells themselves or in stromal and immune cells in the tumour microenvironment (TME). cGAS-STING signalling results in the production of type I interferons, which have been linked to immune cell infiltration in 'hot' tumours that are susceptible to immunosurveillance and immunotherapy approaches. However, recent research has highlighted the complex nature of STING signalling, with tumours having developed mechanisms to evade and hijack this signalling pathway for their own benefit. In this mini-review we will explore how cGAS-STING signalling in different cells in the TME can promote both anti-tumour and pro-tumour responses. This includes the role of type I interferons and the second messenger cGAMP in the TME, and the influence of STING signalling on local immune cell populations. We examine how alternative signalling cascades downstream of STING can promote chronic interferon signalling, the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the production of inflammatory cytokines, which can have pro-tumour functions. An in-depth understanding of DNA sensing in different cell contexts will be required to harness the anti-tumour functions of STING signalling.
Topics: Humans; Immunity, Innate; DNA; Nucleotidyltransferases; Interferon Type I; Neoplasms; Tumor Microenvironment
PubMed: 37534795
DOI: 10.1042/EBC20220241 -
BMC Genomics Jul 2020Previous studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA...
BACKGROUND
Previous studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; however, the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application.
RESULTS
Using targeted sequencing of cfDNA in a large cohort of solid tumor patient, sequencing reads harboring tumor-specific somatic mutations were isolated to examine the exact size distribution of tumor cfDNA. For the majority of studied cases, 166 bp remained as the peak size of tumor cfDNA, with tumor cfDNA showing an increased proportion of short fragments (100-150 bp). Less than 1% of cfDNA samples were found to be peaked at 134/144 bp and independent of tumor cfDNA purity. Using whole-genome sequencing of cfDNA, we discovered a positive correlation between cfDNA shortening and the magnitude of chromatin inaccessibility, as measured by transcription, DNase I hypersensitivity, and histone modifications. Tumor cfDNA shortening occurred simultaneously at both 5' and 3' ends of the DNA wrapped around nucleosomes.
CONCLUSIONS
Tumor cfDNA shortening exhibited two distinctive modes. Tumor cfDNA purity and chromatin inaccessibility were contributing factors but insufficient to trigger a global transition from 166 bp dominant to 134/144 bp dominant phenotype.
Topics: Biomarkers, Tumor; Chromatin Assembly and Disassembly; Circulating Tumor DNA; DNA Fragmentation; Female; Humans; Male; Middle Aged; Mutation; Neoplasms; Nucleosomes; Whole Genome Sequencing
PubMed: 32650715
DOI: 10.1186/s12864-020-06848-9 -
Journal of Cancer Research and Clinical... Sep 2023The role of the epithelial cell adhesion molecule (EpCAM) in cancer is still unclear. EpCAM cleavage through regulated intramembrane proteolysis results in fragments...
BACKGROUND
The role of the epithelial cell adhesion molecule (EpCAM) in cancer is still unclear. EpCAM cleavage through regulated intramembrane proteolysis results in fragments which interact with both oncogenic and tumor suppressive pathways. Additionally, the EpCAM molecule itself is used as a descriptive therapeutic target in urothelial cancer (UC), while data on its actual tumor specificity remain limited.
METHODS
Samples from diagnostic formalin-fixed paraffin-embedded (FFPE) UC tissue and fresh-frozen UC cells were immunoblotted and used for qualitative characterization of five different EpCAM fragments. These expression patterns were quantified across a cohort of 76 samples with 52 UC and 24 normal urothelial samples. Cell viability effects of the extracellular EpEX fragment were assessed in the UC cell lines T24 and HT1376.
RESULTS
The proteolytic EpCAM fragments could be identified in clinical FFPE tissue specimens too. Neither overall nor fragment-specific EpCAM expression showed relevant tumor specificity. EpEX and its deglycosylated variant showed an inverse relationship across healthy and tumor tissue with a decrease of deglycosylated EpEX in tumors. However, extracellular EpEX did not show a relevant effect in vitro.
CONCLUSIONS
EpCAM should not be regarded as tumor-specific in UC without patient-specific predictive testing. EpCAM fragment patterns indicate cancer-specific changes and could be involved in its complex tumor-biological role.
Topics: Humans; Epithelial Cell Adhesion Molecule; Cell Adhesion Molecules; Antigens, Neoplasm; Carcinoma, Transitional Cell; Urinary Bladder Neoplasms; Cell Line, Tumor
PubMed: 37154925
DOI: 10.1007/s00432-023-04809-9 -
Theranostics 2023Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human... (Review)
Review
Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness . To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies.
Topics: Humans; Immunoglobulin Fragments; Antigens, Tumor-Associated, Carbohydrate; Antibodies, Monoclonal; Neoplasms; Polysaccharides; Tumor Microenvironment
PubMed: 37284439
DOI: 10.7150/thno.80901 -
Human Genomics Oct 2023Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in...
BACKGROUND
Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in the field of liquid biopsy. However, the clinical application of this approach has been hindered by a limited understanding of cfDNA biology. Furthermore, the prevalence of hematopoietic cell-derived cfDNA in plasma complicates the in vivo investigation of tissue-specific cfDNA other than that of hematopoietic origin. While conventional two-dimensional cell lines have contributed to research on cfDNA biology, their limited representation of in vivo tissue contexts underscores the need for more robust models. In this study, we propose three-dimensional organoids as a novel in vitro model for studying cfDNA biology, focusing on multifaceted fragmentomic analyses.
RESULTS
We established nine patient-derived organoid lines from normal lung airway, normal gastric, and gastric cancer tissues. We then extracted cfDNA from the culture medium of these organoids in both proliferative and apoptotic states. Using whole-genome sequencing data from cfDNA, we analyzed various fragmentomic features, including fragment size, footprints, end motifs, and repeat types at the end. The distribution of cfDNA fragment sizes in organoids, especially in apoptosis samples, was similar to that found in plasma, implying occupancy by mononucleosomes. The footprints determined by sequencing depth exhibited distinct patterns depending on fragment sizes, reflecting occupancy by a variety of DNA-binding proteins. Notably, we discovered that short fragments (< 118 bp) were exclusively enriched in the proliferative state and exhibited distinct fragmentomic profiles, characterized by 3 bp palindromic end motifs and specific repeats.
CONCLUSIONS
In conclusion, our results highlight the utility of in vitro organoid models as a valuable tool for studying cfDNA biology and its associated fragmentation patterns. This, in turn, will pave the way for further enhancements in noninvasive cancer detection methodologies based on fragmentomics.
Topics: Humans; Cell-Free Nucleic Acids; Neoplasms; Liquid Biopsy; Whole Genome Sequencing; Cell Line; Biomarkers, Tumor
PubMed: 37898819
DOI: 10.1186/s40246-023-00533-0