AZD7762 induces CRBN dependent BAG3 degradation through ubiquitin-proteasome pathway.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

miR-198 inhibits the progression of renal cell carcinoma by targeting BIRC5.

BACKGROUND: miR-198 is involved in the formation, migration, invasion, and metastasis of various malignant cancers. However, the function and mechanism of action of miR-198 in the tumorigenesis of renal cell carcinoma (RCC) remain elusive. Here, we aimed to explore the role of miR198 in RCC. METHODS: Immunohistochemistry was performed to estimate the level of survivin in RCC sections. Quantitative real-time polymerase chain reaction was performed to determine the expression level of miR-198 in fresh RCC tissues. Furthermore, the target relationship between miR-198 and BIRC5 was predicted using the TargetScanHuman 7.2 database and verified via dual-luciferase reporter assay and western blotting. The effects of miR-198 on the viability, apoptosis, invasion, and migration of A498 and ACHN cells were studied using Cell Counting Kit-8, flow cytometry, transwell migration assay, and wound healing assay, respectively. Additionally, a xenograft nude mouse model was established to evaluate the effect of miR-198 on RCC tumorigenesis. RESULTS: The expression levels of BIRC5 and miR-198 were respectively higher and lower in RCC tissues than those in normal adjacent tissues. Furthermore, miR-198 could inhibit luciferase activity and reduce the protein level of survivin without affecting the BIRC5 mRNA levels. miR-198 inhibited cell viability, migration, and invasion and promoted cell apoptosis; co-transfection with BIRC5 could rescue these effects. Moreover, miR-198 could repress tumor growth in the xenograft nude mouse model of RCC. CONCLUSIONS: Our study demonstrates that miR-198 suppresses RCC progression by targeting BIRC5.

Mutation in NPPA causes atrial fibrillation by activating inflammation and cardiac fibrosis in a knock-in rat model.

Atrial fibrillation (AF) affects >30 million individuals worldwide. However, no genetic mutation from human patients with AF has been linked to inflammation. Here, we show that AF-associated human variant p.Ile138Thr in natriuretic peptide A (NPPA) encoding the atrial natriuretic peptide (ANP) causes inflammation, fibroblast activation, atrial fibrosis, and AF in knock-in (KI) rats. Variant p.Ile138Thr inhibits the interaction between ANP and its receptor natriuretic peptide receptor A and reduces intracellular cGMP levels. RNA sequencing and follow-up analyses showed that mutant ANP (mANP) activates multiple innate immunity pathways, including TNF-α, NF-κB, and IL-1ß signaling. mANP induces differentiation of cardiac fibroblasts (CFs) to myofibroblasts and promotes CF proliferation and fibrosis. These results suggest that NPPA variant p.Ile138Thr causes AF by activating TNF-α, NF-κB, and IL-1ß signaling, inflammation, and fibrosis. Multiple computational programs suggest that p.Ile138Thr is damaging or deleterious. Based on the 2015 American College of Medical Genetics and Genomics Standards and Guidelines, p.Ile138Thr can be classified as a likely pathogenic variant. Variant p.Ile138Thr was found only in Asian people in the Genome Aggregation Database and Exome Aggregation Consortium database at an averaged frequency of 0.026%. An estimated 1.15 million Asian people carry the variant and might be at risk of AF. The KI rats may provide an inflammation-based, genetic animal model for AF valuable for testing anti-inflammation or other therapies for AF.-Cheng, C., Liu, H., Tan, C., Tong, D., Zhao, Y., Liu, X., Si, W., Wang, L., Liang, L., Li, J., Wang, C., Chen, Q., Du, Y., Wang, Q. K., Ren, X. Mutation in NPPA causes atrial fibrillation by activating inflammation and cardiac fibrosis in a knock-in rat model.

Elevated MTA1 induced the migration and invasion of renal cell carcinoma through the NF-κB pathway.

BACKGROUND: The metastasis-associated gene 1 (MTA1) has been extensively reported as a crucial oncogene, and its abnormal expression has been associated with the progression of numerous cancers. However, the role of MTA1 in renal cell carcinoma (RCC) progression and metastasis remains unclear. Herein, we investigated the expression of MTA1 and its role in RCC. METHODS: 109 matched clear cell RCCs (ccRCCs) and corresponding normal tissue samples were analyzed via immunohistochemistry to test the expression of MTA1. Human A498 cell lines were transfected with pcDNA3.1-Flag (control) or Flag-MTA1 to overexpress MTA1 or with specific interfering RNA (si-MTA1) or specific interfering negative control to knockdown MTA1 expression. Transfected cells were used in wound healing and transwell invasion assay. Quantitative real time polymerase chain reaction was used to assess the effect of MTA1 on MMP2/MMP9 and E-cadherin gene expression. Western blot was used to qualify the phosphorylation of p65. RESULTS: Herein, we found a significantly increased expression of MTA1 in 109 ccRCCs, compared to the corresponding normal tissue. In addition, the overexpression of MTA1 in A498 cells facilitated cell migration and invasion, while the down-regulation of MTA1 expression using specific interfering RNA sequences could decrease cell migration and invasion. Furthermore, we showed that MTA1 is up-regulated in ccRCCs, which contributes to the migration and invasion of human kidney cancer cells by mediating the expression of MMP2 and MMP9 through the NF-κB signaling pathway. Similarly, we found that MTA1 could regulate E-cadherin expression in RCCs. CONCLUSIONS: MTA1 is overexpressed in RCC and is involved in the progression of RCC through NF-κB.

Angiotensin II increases angiogenesis by NF-κB-mediated transcriptional activation of angiogenic factor AGGF1.

Angiogenic factor with G-patch and FHA domains 1 (AGGF1) is involved in vascular development, angiogenesis, specification of hemangioblasts, and differentiation of veins. When mutated, however, it causes Klippel-Trenaunay syndrome, a vascular disorder. In this study, we show that angiotensin II (AngII)-the major effector of the renin-angiotensin system and one of the most important regulators of the cardiovascular system-induces the expression of AGGF1 through NF-κB, and that AGGF1 plays a key role in AngII-induced angiogenesis. AngII significantly up-regulated the levels of AGGF1 mRNA and protein in HUVECs at concentrations of 10-40 µg/ml but not >60 µg/ml. AngII type 1 receptor (AT1R) inhibitor losartan inhibited AngII-induced up-regulation of AGGF1, whereas AT2R inhibitor PD123319 further increased AngII-induced up-regulation of AGGF1. Up-regulation of AGGF1 by AngII was blocked by NF-κB inhibitors, and p65 binds directly to a binding site at the promoter/regulatory region of AGGF1 and transcriptionally activates AGGF1 expression. AngII-induced endothelial tube formation was blocked by small interfering RNAs (siRNAs) for RELA (RELA proto-oncogene, NF-κB subunit)/p65 or AGGF1, and the effect of RELA siRNA was rescued by AGGF1. AngII-induced angiogenesis from aortic rings was severely impaired in Aggf1+/- mice, and the effect was restored by AGGF1. These data suggest that AngII acts as a critical regulator of AGGF1 expression through NF-κB, and that AGGF1 plays a key role in AngII-induced angiogenesis.-Si, W., Xie, W., Deng, W., Xiao, Y., Karnik, S. S., Xu, C., Chen, Q., Wang, Q. K. Angiotensin II increases angiogenesis by NF-κB-mediated transcriptional activation of angiogenic factor AGGF1.

Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.

Triptolide inhibits the progression of Glioblastoma U251 cells via targeting PROX1.

Background: Glioblastoma multiforme (GBM) is the most lethal brain cancer in adults, characterized by rapid growth, extensive invasiveness, and poor prognosis, and there is still a lack of effective treatments. Here, we aimed to explore the role of triptolide (TPL), purified from Tripterygium wilfordii Hook F, on glioblastoma cell growth, apoptosis, proliferation, migration and invasion, as well as potential underlying mechanisms. Methods: The publicly available clinical data of Brain Lower Grade Glioma (LGG) from The Cancer Genome Atlas (TCGA) had been screened to observe PROX1 expression. The Kaplan-Meier analysis was used to analyze the relationship between PROX1 expression and GBM prognosis. CCK8, cell cycle, EDU, apoptosis, wound healing, and transwell assays were performed to detect the effects of TPL on glioblastoma U251 cell viability, cell cycle, proliferation, apoptosis, migration and invasion, respectively. Further, a soft agar colony assay was used to calculate the growth of glioblastoma cells. The qRT-PCR and western blot were conducted to quantify PROX1 mRNA and protein levels. The transcriptional regulation of TPL was detected by Dual luciferase reporter assay. Results: We found that TPL inhibited glioblastoma cell viability, proliferation, cell cycle, migration and invasion, but enhanced apoptosis in a dose-dependent manner. The expression of cell cycle inhibitor, P21, and pro-apoptosis factor, Bax was increased, while invasion-related factors MMP2 and MMP9 were silenced after TPL treatments. Mechanistically, TPL showed transcriptional inhibition of PROX1 appearance. Moreover, ectopic expression of PROX1 partially rescued the effects of TPL on glioblastoma cell viability, proliferation, apoptosis, migration and invasion, and on the expression of cell function-related genes. Conclusion: This study verified that TPL inhibited the progression of glioblastoma cells by transcriptionally depressing the expression of PROX1.

[Network pharmacology-based study of the therapeutic mechanism of resveratrol for Alzheimer's disease].

OBJECTIVE: To investigate the therapeutic mechanism of resveratrol (RES) for Alzheimer's disease (AD) in light of network pharmacology. METHODS: We searched PubChem, BATMAN-TCM, Genecards, AD, TTD, String 11.0, AlzData, SwissTargetPrediction, Metascape and other databases for the therapeutic targets of RES and human AD-related targets. The intersection was determined using Venny 2.1 to obtain the therapeutic targets of RES for AD. The protein-protein interaction (PPI) network was constructed, the gene ontology (GO) was enriched and the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) were analyzed. Cytoscape 3.7.1 software was used to construct a target-signaling pathway network of RES in the treatment of AD. Molecular docking verification was carried out on SwissDock (http://www.swissdock.ch/docking). We examined a 293Tau cell model of AD for changes in protein levels of pS396, pS199, Tau5, CDK5, glycogen synthase kinase 3ß (GSK3ß) and p-GSK3ß in response to RES treatment using Western blotting. RESULTS: We obtained 182 targets of RES, 525 targets related to AD, and 36 targets of RES for AD treatment, among which 34.6% of the targets were protein-modifying enzymes, 27.7% were metabolite invertase, 13.8% were gene-specific transcriptional regulators, and 10.3% were transporters. The core key targets of RES in the treatment of AD included INS, APP, ESR1, MMP9, IGF1R, CACNA1C, MAPT (microtubule- associated protein Tau), MMP2, TGFB1 and GSK3B. Enrichment analysis of GO biological process suggested that the biological function of RES in AD treatment mainly involved the response to ß-amyloid protein, positive regulation of transferase activity, the transmembrane receptor protein tyrosine kinase signaling pathway, regulation of behavior, learning or memory, aging, and transmembrane transport. KEGG pathway enrichment analysis showed that the most significantly enriched signaling pathways were AD pathway, PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, and MAPK signaling pathway. Molecular docking results showed that RES had strong binding with ESR1, GSK3B, MMP9, IGF1R, APP and INS. In the cell model of AD, treatment with 50 µmol/L RES for 12 h significantly reduced the levels of pS396 and pS199 by regulating CDK5 and GSK3ß activity (P < 0.001). CONCLUSIONS: RES produces therapeutic effects on AD by acting on multiple targets and affecting multiple signaling pathways and improves AD-associated pathologies via a direct action on Aß and Tau pathological processes.

Angiogenic factor AGGF1 acts as a tumor suppressor by modulating p53 post-transcriptional modifications and stability via MDM2.

Angiogenesis factors are widely known to promote tumor growth by increasing tumor angiogenesis in the tumor microenvironment, however, little is known whether their intracellular function is involved in tumorigenesis. Here we show that AGGF1 acts as a tumor suppressor by regulating p53 when acting inside tumor cells. AGGF1 antagonizes MDM2 function to inhibit p53 ubiquitination, increases the acetylation, phosphorylation, stability and expression levels of p53, activates transcription of p53 target genes, and regulates cell proliferation, cell cycle, and apoptosis. AGGF1 also interacts with p53 through the FHA domain. Somatic AGGF1 variants in the FHA domain in human tumors, including p.Q467H, p.Y469 N, and p.N483T, inhibit AGGF1 activity on tumor suppression. These results identify a key role for AGGF1 in an AGGF1-MDM2-p53 signaling axis with important functions in tumor suppression, and uncover a novel trans-tumor-suppression mechanism dependent on p53. This study has potential implications in diagnosis and therapies of cancer.

MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development.

Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hbαe1, hbße1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3'-UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1.

Transcriptional activation of the Prox1 gene by HIF-1α and HIF-2α in response to hypoxia.

Prox1 encodes a homeobox transcription factor critical to organ development, but its regulation is poorly understood. Here, we show that Prox1 expression is induced by hypoxia, and controlled by a hypoxia-response element (HRE) at the Prox1 promoter/regulatory region and HIF-1α/HIF-2α. EMSA and ChIP assays demonstrated the direct interaction of the HRE with HIF-1α or HIF-2α. Overexpression of HIF-1α or HIF-2α increased activation of the Prox1 promoter, whereas knockdown of HIF-1α or HIF-2α inhibited the activation. These data reveal a novel molecular mechanism for regulation of Prox1 expression in response to hypoxia and provide new insights into Prox1-controlled processes such as lymphangiogenesis.

MicroRNA-503 targets FGF2 and VEGFA and inhibits tumor angiogenesis and growth.

FGF2 and VEGFA are the two most potent angiogenic factors. Here we report that miR-503 can simultaneously down-regulate FGF2 and VEGFA. The expression of miR-503 is repressed in HCC cells and primary tumors due to a potential epigenetic mechanism. Overexpression of miR-503 reduced tumor angiogenesis in vitro and in vivo. We also found that miR-503 expression was down-regulated by hypoxia through HIF1α. These results identify a miRNA that targets both FGF2 and VEGFA in cancers, demonstrate the anti-angiogenesis role of miR-503 in tumorigenesis, and provide a novel mechanism for hypoxia-induced FGF2 and VEGFA through HIF1α-mediated inhibition of miR-503.