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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 -
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 -
European Journal of Medicinal Chemistry Dec 2023Targeted protein degradation (TPD) has emerged as a promising therapeutic approach with potential advantages over traditional occupancy-based inhibitors in terms of... (Review)
Review
Targeted protein degradation (TPD) has emerged as a promising therapeutic approach with potential advantages over traditional occupancy-based inhibitors in terms of dosing, side effects and targeting "undruggable" proteins. Targeted degraders can theoretically bind any nook or cranny of targeted proteins to drive degradation. This offers convenience versus the small-molecule inhibitors that must function in a well-defined pocket. The degradation process depends mainly on two cell self-destruction mechanisms, namely the ubiquitin-proteasome system and the lysosomal degradation pathway. Various TPD strategies (e.g., proteolytic-targeting chimeras, molecular glues, lysosome-targeting chimeras, and autophagy-targeting chimeras) have been developed. These approaches hold great potential for targeting dysregulated proteins, potentially offering therapeutic benefits. In this article, we systematically review the mechanisms of various TPD strategies, potential applications to drug discovery, and recent advances. We also discuss the benefits and challenges associated with these TPD strategies, aiming to provide insight into the targeting of dysregulated proteins and facilitate their clinical applications.
Topics: Proteolysis; Proteasome Endopeptidase Complex; Autophagy; Drug Discovery; Lysosomes
PubMed: 37778240
DOI: 10.1016/j.ejmech.2023.115839 -
Gene Apr 2024Breast cancer (BC) is a highly frequent malignant tumor that poses a serious threat to women's health and has different molecular subtypes, histological subtypes, and... (Review)
Review
Breast cancer (BC) is a highly frequent malignant tumor that poses a serious threat to women's health and has different molecular subtypes, histological subtypes, and biological features, which act by activating oncogenic factors and suppressing cancer inhibitors. The ubiquitin-proteasome system (UPS) is the main process contributing to protein degradation, and deubiquitinases (DUBs) are reverse enzymes that counteract this process. There is growing evidence that dysregulation of DUBs is involved in the occurrence of BC. Herein, we review recent research findings in BC-associated DUBs, describe their nature, classification, and functions, and discuss the potential mechanisms of DUB-related dysregulation in BC. Furthermore, we present the successful treatment of malignant cancer with DUB inhibitors, as well as analyzing the status of targeting aberrant DUBs in BC.
Topics: Humans; Female; Breast Neoplasms; Antineoplastic Agents; Ubiquitin; Proteasome Endopeptidase Complex; Cytoplasm; Ubiquitination
PubMed: 38242375
DOI: 10.1016/j.gene.2024.148175 -
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 FEBS Journal Dec 2023Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a...
Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK), and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.
Topics: Humans; Proteasome Endopeptidase Complex; Proteostasis; HEK293 Cells; Molecular Chaperones; Unfolded Protein Response; Endoplasmic Reticulum Stress; Endoplasmic Reticulum Chaperone BiP; Carrier Proteins; Peptides
PubMed: 37665644
DOI: 10.1111/febs.16948 -
Journal of Medicinal Chemistry Nov 2023Protein tyrosine phosphatase nonreceptor Type 2 (PTPN2) is an attractive target for cancer immunotherapy. PTPN2 and another subtype of PTP1B are highly similar in...
Protein tyrosine phosphatase nonreceptor Type 2 (PTPN2) is an attractive target for cancer immunotherapy. PTPN2 and another subtype of PTP1B are highly similar in structure, but their biological functions are distinct. Therefore, subtype-selective targeting of PTPN2 remains a challenge for researchers. Herein, the development of small molecular PTPN2 degraders based on a thiadiazolidinone dioxide-naphthalene scaffold and a VHL E3 ligase ligand is described, and the PTPN2/PTP1B subtype-selective degradation is achieved for the first time. The linker structure modifications led to the discovery of the subtype-selective PTPN2 degrader (PTPN2/PTP1B selective index > 60-fold), which also exhibits excellent proteome-wide degradation selectivity. induces PTPN2 degradation in a ubiquitination- and proteasome-dependent manner. It efficiently promotes T cell activation and amplifies IFN-γ-mediated B16F10 cell growth inhibition. This study provides a convenient chemical knockdown tool for PTPN2-related research and a paradigm for subtype-selective PTP degradation through nonspecific substrate-mimicking ligands, demonstrating the therapeutic potential of PTPN2 subtype-selective degradation.
Topics: Protein Tyrosine Phosphatase, Non-Receptor Type 2; Phosphorylation; Ubiquitin-Protein Ligases; Ubiquitination; Proteasome Endopeptidase Complex
PubMed: 37966047
DOI: 10.1021/acs.jmedchem.3c01348 -
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 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Dec 2023Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules by utilizing the ubiquitin proteasome system (UPS) to degrade proteins of interest.... (Review)
Review
Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules by utilizing the ubiquitin proteasome system (UPS) to degrade proteins of interest. PROTACs have exhibited unprecedented efficacy and specificity in degrading various oncogenic proteins because of their unique mechanism of action, ability to target "undruggable" and mutant proteins. A series of PROTACs have been developed to degrade multiple key protein targets for the treatment of hematologic malignancy. Notably, PROTACs that target BCL-XL, IRAK4, STAT3 and BTK have entered clinical trials. The known PROTACs that have the potential to be used to treat various hematological malignancies are systematically summarized in this review.
Topics: Humans; Hematologic Neoplasms; Proteasome Endopeptidase Complex; Ubiquitin-Protein Ligases; Proteolysis Targeting Chimera
PubMed: 38071084
DOI: 10.19746/j.cnki.issn.1009-2137.2023.06.051