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Nature Communications Sep 2023In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting...
In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.
Topics: Autophagy; Autophagosomes; Cytosol; Macroautophagy; Ribosomes
PubMed: 37726301
DOI: 10.1038/s41467-023-41525-x -
Acta Pharmaceutica Sinica. B Oct 2023The ubiquitin-proteasome system (UPS) dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms. These enzymatic cascades mark... (Review)
Review
The ubiquitin-proteasome system (UPS) dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms. These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules. In the UPS, E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis. As the major regulators of protein homeostasis, E3 ligases are indispensable to proper cell manners in diverse systems, and they are well described in physiological bone growth and bone metabolism. Pathologically, classic bone-related diseases such as metabolic bone diseases, arthritis, bone neoplasms and bone metastasis of the tumor, etc., were also depicted in a UPS-dependent manner. Therefore, skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism. Furthermore, based on the current status of treatment, normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research. As a strategy possibly remedies the limitations of UPS treatment, emerging PROTAC was described comprehensively to illustrate its potential in clinical application. Altogether, the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.
PubMed: 37799379
DOI: 10.1016/j.apsb.2023.06.016 -
Cell Death & Disease Aug 2023Metabolism vulnerability of cisplatin resistance in BCa cells remains to be discovered, which we applied integrated multi-omics analysis to elucidate the metabolism...
Metabolism vulnerability of cisplatin resistance in BCa cells remains to be discovered, which we applied integrated multi-omics analysis to elucidate the metabolism related regulation mechanism in bladder cancer (BCa) microenvironment. Integrated multi-omics analysis of metabolomics and proteomics revealed that MAT2A regulated methionine metabolism contributes to cisplatin resistance in BCa cells. We further validated MAT2A and cancer stem cell markers were up-regulated and circARHGAP10 was down-regulated through the regulation of MAT2A protein stability in cisplatin resistant BCa cells. circARHGAP10 formed a complex with MAT2A and TRIM25 to accelerate the degradation of MAT2A through ubiquitin-proteasome pathway. Knockdown of MAT2A through overexpression of circARHGAP10 and restriction of methionine up-take was sufficient to overcome cisplatin resistance in vivo in immuno-deficiency model but not in immuno-competent model. Tumor-infiltrating CD8 T cells characterized an exhausted phenotype in tumors with low methionine. High expression of SLC7A6 in BCa negatively correlated with expression of CD8. Synergistic inhibition of MAT2A and SLC7A6 could overcome cisplatin resistance in immuno-competent model in vivo. Cisplatin resistant BCa cells rely on methionine for survival and stem cell renewal. circARHGAP10/TRIM25/MAT2A regulation pathway plays an important role in cisplatin resistant BCa cells while circARHGAP10 and SLC7A6 should be evaluated as one of the therapeutic target of cisplatin resistant BCa.
Topics: Humans; Urinary Bladder Neoplasms; Tumor Microenvironment; Methionine; Proteomics; Metabolomics; Cisplatin; Neoplastic Stem Cells; RNA, Circular; Ubiquitin; Proteasome Endopeptidase Complex; Ubiquitination; Drug Resistance, Neoplasm; Animals; Mice; Mice, Nude; Mice, Inbred BALB C; CD8-Positive T-Lymphocytes
PubMed: 37582769
DOI: 10.1038/s41419-023-06050-1 -
Cancer Research Aug 2023Liposarcoma is the most commonly occurring soft-tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes...
UNLABELLED
Liposarcoma is the most commonly occurring soft-tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes MDM2 and CDK4. This unique genetic profile makes liposarcoma an attractive candidate for targeted therapeutics. While CDK4/6 inhibitors are currently employed for treatment of several cancers, MDM2 inhibitors have yet to attain clinical approval. Here, we report the molecular characterization of the response of liposarcoma to the MDM2 inhibitor nutlin-3. Treatment with nutlin-3 led to upregulation of two nodes of the proteostasis network: the ribosome and the proteasome. CRISPR/Cas9 was used to perform a genome-wide loss of function screen that identified PSMD9, which encodes a proteasome subunit, as a regulator of response to nutlin-3. Accordingly, pharmacologic studies with a panel of proteasome inhibitors revealed strong combinatorial induction of apoptosis with nutlin-3. Mechanistic studies identified activation of the ATF4/CHOP stress response axis as a potential node of interaction between nutlin-3 and the proteasome inhibitor carfilzomib. CRISPR/Cas9 gene editing experiments confirmed that ATF4, CHOP, and the BH3-only protein, NOXA, are all required for nutlin-3 and carfilzomib-induced apoptosis. Furthermore, activation of the unfolded protein response using tunicamycin and thapsigargin was sufficient to activate the ATF4/CHOP stress response axis and sensitize to nutlin-3. Finally, cell line and patient-derived xenograft models demonstrated combinatorial effects of treatment with idasanutlin and carfilzomib on liposarcoma growth in vivo. Together, these data indicate that targeting of the proteasome could improve the efficacy of MDM2 inhibitors in liposarcoma.
SIGNIFICANCE
Targeting the proteasome in combination with MDM2 inhibition activates the ATF4/CHOP stress response axis to induce apoptosis in liposarcoma, providing a potential therapeutic approach for the most common soft-tissue sarcoma.
Topics: Humans; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Liposarcoma; Antineoplastic Agents; Proteasome Inhibitors; Apoptosis; Activating Transcription Factor 4
PubMed: 37205634
DOI: 10.1158/0008-5472.CAN-22-3173 -
Journal of Experimental & Clinical... Nov 2023TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in...
BACKGROUND
TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape.
METHODS
In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections.
RESULTS
A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland.
CONCLUSIONS
We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.
Topics: Animals; Female; Humans; Mice; Breast Neoplasms; Epithelial-Mesenchymal Transition; Mammary Neoplasms, Animal; Proteasome Endopeptidase Complex; Proteomics; Signal Transduction; Transforming Growth Factor beta
PubMed: 38008756
DOI: 10.1186/s13046-023-02909-5 -
International Journal of Biological... 2024A growing number of studies have revealed an association between proteasome activator complex subunit 2 (PSME2) and the progression of various forms of cancer. However,...
A growing number of studies have revealed an association between proteasome activator complex subunit 2 (PSME2) and the progression of various forms of cancer. However, the effect of PSME2 on osteosarcoma progression is unknown. Pan-cancer analyses focused on the immunological activity and prognostic relevance of PSME2 have yet to be conducted. The Cancer Genome Atlas and Genome-Tissue Expression databases were leveraged to evaluate PSME2 expression and activity across 33 cancer types. Significant PSME2 dysregulation was noted in a wide range of cancer types and this gene was found to offer significant diagnostic and prognostic utility in most analyzed cancers. From a mechanistic perspective, PSME2 expression levels were correlated with DNA methylation, DNA repair, genomic instability, and TME scores in multiple cancer types. PSME2 was subsequently established as a pan-cancer biomarker of M1 macrophage infiltration based on a combination of bulk, single-cell, and spatial transcriptomic data and confirmatory fluorescent staining results. In osteosarcoma cells, overexpressing PSME2 significantly suppressed tumor proliferative, migratory, and invasive activity. Screening efforts also successfully identified the PSME2-activating drug irinotecan, which can synergistically promote the death of osteosarcoma cells when combined with the chemotherapeutic drug paclitaxel. As a biomarker of M1 macrophage infiltration, PSME2 expression levels may offer insight into tumor development and progression for a wide range of cancers including osteosarcoma, emphasizing its potential utility as a prognostic and therapeutic target worthy of further study.
Topics: Humans; Osteosarcoma; Phenotype; Biomarkers, Tumor; Macrophages; Bone Neoplasms; Proteasome Endopeptidase Complex
PubMed: 38385075
DOI: 10.7150/ijbs.90226 -
Experimental & Molecular Medicine Oct 2023High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC...
High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC have been well characterized. However, the patterns of epigenetic alterations and transcription factor dysregulation in HGSOC have not yet been fully elucidated. In this study, we performed integrative omics analyses of a series of stepwise HGSOC model cells originating from human fallopian tube secretory epithelial cells (HFTSECs) to investigate early epigenetic alterations in HGSOC tumorigenesis. Assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) methods were used to analyze HGSOC samples. Additionally, protein expression changes in target genes were confirmed using normal HFTSECs, serous tubal intraepithelial carcinomas (STICs), and HGSOC tissues. Transcription factor motif analysis revealed that the DNA-binding activity of the AP-1 complex and GATA family proteins was dysregulated during early tumorigenesis. The protein expression levels of JUN and FOSL2 were increased, and those of GATA6 and DAB2 were decreased in STIC lesions, which were associated with epithelial-mesenchymal transition (EMT) and proteasome downregulation. The genomic region around the FRA16D site, containing a cadherin cluster region, was epigenetically suppressed by oncogenic signaling. Proteasome inhibition caused the upregulation of chemokine genes, which may facilitate immune evasion during HGSOC tumorigenesis. Importantly, MEK inhibitor treatment reversed these oncogenic alterations, indicating its clinical effectiveness in a subgroup of patients with HGSOC. This result suggests that MEK inhibitor therapy may be an effective treatment option for chemotherapy-resistant HGSOC.
Topics: Female; Humans; Ovarian Neoplasms; Proteasome Endopeptidase Complex; Cystadenocarcinoma, Serous; Carcinogenesis; Transcription Factors; Epigenesis, Genetic; Mitogen-Activated Protein Kinase Kinases
PubMed: 37779141
DOI: 10.1038/s12276-023-01090-1 -
Redox Biology Sep 2023The nuclear factor erythroid 2 (NF-E2)-related factor 1 (NFE2L1, also known as Nrf1) is a highly conserved transcription factor that belongs to the CNC-bZIP subfamily.... (Review)
Review
The nuclear factor erythroid 2 (NF-E2)-related factor 1 (NFE2L1, also known as Nrf1) is a highly conserved transcription factor that belongs to the CNC-bZIP subfamily. Its significance lies in its control over redox balance, proteasome activity, and organ integrity. Stress responses encompass a series of compensatory adaptations utilized by cells and organisms to cope with extracellular or intracellular stress initiated by stressful stimuli. Recently, extensive evidence has demonstrated that NFE2L1 plays a crucial role in cellular stress adaptation by 1) responding to oxidative stress through the induction of antioxidative responses, and 2) addressing proteotoxic stress or endoplasmic reticulum (ER) stress by regulating the ubiquitin-proteasome system (UPS), unfolded protein response (UPR), and ER-associated degradation (ERAD). It is worth noting that NFE2L1 serves as a core factor in proteotoxic stress adaptation, which has been extensively studied in cancer and neurodegeneration associated with enhanced proteasomal stress. In these contexts, utilization of NFE2L1 inhibitors to attenuate proteasome "bounce-back" response holds tremendous potential for enhancing the efficacy of proteasome inhibitors. Additionally, abnormal stress adaptations of NFE2L1 and disturbances in redox and protein homeostasis contribute to the pathophysiological complications of cardiovascular diseases, inflammatory diseases, and autoimmune diseases. Therefore, a comprehensive exploration of the molecular basis of NFE2L1 and NFE2L1-mediated diseases related to stress responses would not only facilitate the identification of novel diagnostic and prognostic indicators but also enable the identification of specific therapeutic targets for NFE2L1-related diseases.
Topics: Gene Expression Regulation; NF-E2-Related Factor 1; Oxidative Stress; Proteasome Endopeptidase Complex; Ubiquitin; Humans
PubMed: 37473701
DOI: 10.1016/j.redox.2023.102819 -
Molecular Neurodegeneration May 2024Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and...
Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.
Topics: Animals; Synucleinopathies; alpha-Synuclein; Mice; Humans; Single-Domain Antibodies; Disease Models, Animal; Brain; Proteasome Endopeptidase Complex
PubMed: 38816762
DOI: 10.1186/s13024-024-00730-y -
The Journal of Clinical Investigation Dec 2023Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via...
Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus-bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8-dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.
Topics: Humans; Carcinoma, Hepatocellular; Hepatitis B virus; Liver Neoplasms; Trans-Activators; Proteasome Endopeptidase Complex; Transcription Factors; Hepatitis B; Homologous Recombination; Intracellular Signaling Peptides and Proteins
PubMed: 37815873
DOI: 10.1172/JCI171533