Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Tripathi BK
PROVIDER: S-EPMC8642553 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Medulloblastoma (MB) is a malignant pediatric brain tumor for which new therapies are urgently needed. We demonstrate that treatment with EPZ-6438 (Tazemetostat), an enhancer of zeste homolog 2 (EZH2) inhibitor approved for clinical trials, blocks MB cell growth in vitro and in vivo, and prolongs survival in orthotopic xenograft models. We show that the therapeutic effect is dependent on epigenetic reactivation of adhesion G-protein-coupled receptor B1 (BAI1/ADGRB1), a tumor suppressor that controls p53 stability by blocking Mdm2. Histone 3 trimethylated on lysine 27 (H3K27me3), a marker of silent chromatin conformation is present at the ADGRB1 promoter, and inhibition of EZH2, the catalytic component of the Polycomb Repressive complex 2 (PRC2) that methylates H3K27, switches the gene into an active chromatin status and reactivates BAI1 expression. Mechanistically, targeting EZH2 promotes transition from H3K27me3 to H3K27ac at the promoter, recruits the C/EBPβ (CREB-binding protein) and CBP transcription factors and activates ADGRB1 gene transcription. Taken together, our results identify key molecular players that regulate ADGRB1 gene expression in MB, demonstrate that reactivation of BAI1 expression underlies EPZ-6438 antitumorigenic action, and provide preclinical proof-of-principle evidence for targeting EZH2 in patients with MB.
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Project description:Multiple Myeloma (MM) is a plasma cell tumor localized to the bone marrow (BM). Despite the fact that current treatment strategies have improved patients' median survival time, MM remains incurable. Epigenetic aberrations are emerging as important players in tumorigenesis making them attractive targets for therapy in cancer including MM. Recently, we suggested the polycomb repressive complex 2 (PRC2) as a common denominator of gene silencing in MM and presented the PRC2 enzymatic subunit enhancer of zeste homolog 2 (EZH2) as a potential therapeutic target in MM. Here we further dissect the anti-myeloma mechanisms mediated by EZH2 inhibition and show that pharmacological inhibition of EZH2 reduces the expression of MM-associated oncogenes; IRF-4, XBP-1, PRDM1/BLIMP-1 and c-MYC. We show that EZH2 inhibition reactivates the expression of microRNAs with tumor suppressor functions predicted to target MM-associated oncogenes; primarily miR-125a-3p and miR-320c. ChIP analysis reveals that miR-125a-3p and miR-320c are targets of EZH2 and H3K27me3 in MM cell lines and primary cells. Our results further highlight that polycomb-mediated silencing in MM includes microRNAs with tumor suppressor activity. This novel role strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM.
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Project description:Many newly synthesized cellular proteins pass through the Golgi complex from where secretory transport carriers sort them to the plasma membrane and the extracellular environment. The formation of these secretory carriers at the trans-Golgi network is promoted by the protein kinase D (PKD) family of serine/threonine kinases. Here, using mathematical modeling and experimental validation of the PKD activation and substrate phosphorylation kinetics, we reveal that the expression level of the PKD substrate deleted in liver cancer 1 (DLC1), a Rho GTPase-activating protein that is inhibited by PKD-mediated phosphorylation, determines PKD activity at the Golgi membranes. RNAi-mediated depletion of DLC1 reduced PKD activity in a Rho-Rho-associated protein kinase (ROCK)-dependent manner, impaired the exocytosis of the cargo protein horseradish peroxidase, and was associated with the accumulation of the small GTPase RAB6 on Golgi membranes, indicating a protein-trafficking defect. In summary, our findings reveal that DLC1 maintains basal activation of PKD at the Golgi and Golgi secretory activity, in part by down-regulating Rho-ROCK signaling. We propose that PKD senses cytoskeletal changes downstream of DLC1 to coordinate Rho signaling with Golgi secretory function.
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Project description:The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin-proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.
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