Storax Attenuates Cardiac Fibrosis following Acute Myocardial Infarction in Rats via Suppression of AT1R-Ankrd1-P53 Signaling Pathway.

Myocardial fibrosis following acute myocardial infarction (AMI) seriously affects the prognosis and survival rate of patients. This study explores the role and regulation mechanism of storax, a commonly used traditional Chinese medicine for treatment of cardiovascular diseases, on myocardial fibrosis and cardiac function. The AMI rat model was established by subcutaneous injection of Isoproterenol hydrochloride (ISO). Storax (0.1, 0.2, 0.4 g/kg) was administered by gavage once/d for 7 days. Electrocardiogram, echocardiography, hemodynamic and cardiac enzyme in AMI rats were measured. HE, Masson, immunofluorescence and TUNEL staining were used to observe the degree of pathological damage, fibrosis and cardiomyocyte apoptosis in myocardial tissue, respectively. Expression of AT1R, CARP and their downstream related apoptotic proteins were detected by WB. The results demonstrated that storax could significantly improve cardiac electrophysiology and function, decrease serum cardiac enzyme activity, reduce type I and III collagen contents to improve fibrosis and alleviate myocardial pathological damage and cardiomyocyte apoptosis. It also found that storax can significantly down-regulate expression of AT1R, Ankrd1, P53, P-p53 (ser 15), Bax and cleaved Caspase-3 and up-regulate expression of Mdm2 and Bcl-2. Taken together, these findings indicated that storax effectively protected cardiomyocytes against myocardial fibrosis and cardiac dysfunction by inhibiting the AT1R-Ankrd1-P53 signaling pathway.

Targeted Covalent Inhibition of Small CTD Phosphatase 1 to Promote the Degradation of the REST Transcription Factor in Human Cells.

The repressor element-1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. A high level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small-molecule inhibitors, the inactivation of a regulatory protein, small CTD phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,ß-unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time- and dose-dependent manner with an EC50 ∼1.5 µM, reducing REST protein levels and activating specific REST-suppressed genes. These compounds represent a promising line of small-molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Molecular features of exceptional response to neoadjuvant anti-androgen therapy in high-risk localized prostate cancer.

High-risk localized prostate cancer (HRLPC) is associated with a substantial risk of recurrence and disease mortality. Recent clinical trials have shown that intensifying anti-androgen therapies administered before prostatectomy can induce pathologic complete responses or minimal residual disease, called exceptional response, although the molecular determinants of these clinical outcomes are largely unknown. Here, we perform whole-exome and transcriptome sequencing on pre-treatment multi-regional tumor biopsies from exceptional responders (ERs) and non-responders (NRs, pathologic T3 or lymph node-positive disease) to intensive neoadjuvant anti-androgen therapies. Clonal SPOP mutation and SPOPL copy-number loss are exclusively observed in ERs, while clonal TP53 mutation and PTEN copy-number loss are exclusively observed in NRs. Transcriptional programs involving androgen signaling and TGF-ß signaling are enriched in ERs and NRs, respectively. These findings may guide prospective validation studies of these molecular features in large HRLPC clinical cohorts treated with neoadjuvant anti-androgens to improve patient stratification.

The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation.

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.

Functional and structural characterization of AntR, an Sb(III) responsive transcriptional repressor.

The ant operon of the antimony-mining bacterium Comamonas testosterone JL40 confers resistance to Sb(III). The operon is transcriptionally regulated by the product of the first gene in the operon, antR. AntR is a member of ArsR/SmtB family of metal/metalloid-responsive repressors resistance. We purified and characterized C. testosterone AntR and demonstrated that it responds to metalloids in the order Sb(III) = methylarsenite (MAs(III) >> As(III)). The protein was crystallized, and the structure was solved at 2.1 Å resolution. The homodimeric structure of AntR adopts a classical ArsR/SmtB topology architecture. The protein has five cysteine residues, of which Cys103a from one monomer and Cys113b from the other monomer, are proposed to form one Sb(III) binding site, and Cys113a and Cys103b forming a second binding site. This is the first report of the structure and binding properties of a transcriptional repressor with high selectivity for environmental antimony.

Resveratrol improves Gly-LDL-induced vascular endothelial cell apoptosis, inflammatory factor secretion and oxidative stress by regulating miR-142-3p and regulating SPRED2-mediated autophagy.

BACKGROUND: Resveratrol improves cell apoptosis and tissue damage induced by high glucose, but the specific mechanism is unknown. METHODS: This is a basic research. We performed cell transfection, real-time fluorescence quantitative PCR (qPCR), flow cytometry, immunofluorescence, western blot, enzyme linked immunosorbent assay (ELISA) and cell viability assay to analyze cell viability, cell cycle, cellular oxidative stress, intracellular inflammatory factors and autophagy activities in vitro. Meanwhile, dual luciferase reporter assay was conducted to explore the influence of miR-142-3p and sprouty-related EVH1 domain 2 (SPRED 2) on human glycated low-density lipoprotein (Gly-LDL)-induced vascular endothelial cell apoptosis, inflammatory factor secretion and oxidative stress. RESULTS: Resveratrol inhibited the expression of miR-142-3p in human umbilical vein endothelial cells (HUVECs) induced by Gly-LDL in a dose-dependent manner, and the overexpression of miR-142-3p reverses the effect of resveratrol on the proliferation, apoptosis, secretion of inflammatory factors, oxidative stress, and autophagy. The dual-luciferase report analysis found a negative regulatory relationship between miR-142-3p and SPRED2. Inhibition of SPRED2 reversed the effects of resveratrol on Gly-LDL-induced HUVECs proliferation, apoptosis, inflammatory factor secretion and oxidative stress, and reversed the effects of resveratrol on Gly-LDL-induced HUVECs autophagy. CONCLUSION: miR-142-3p promotes the development of diabetes by inhibiting SPRED2-mediated autophagy, including inducing cell apoptosis, aggravating cellular oxidative stress and secretion of inflammatory factors, and resveratrol improves this effect.

Discovery of Small-Molecule Antagonists of the PWWP Domain of NSD2.

Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein-protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a Kd of 3.4 µM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.

Synthesis, docking study and inhibitory activity of 2,6-diketopiperazines derived from α-amino acids on HDAC8.

Diketopiperazines (DKPs) have been regarded as an important scaffold from the viewpoint of synthesis due to their biological properties for the treatment of several diseases, including cancer. In this work, two novel series of enantiomeric 2,6-DKPs derived from α-amino acids were synthesized through nucleophilic substitution and intramolecular cyclization reactions. All the compounds were docked against histone deacetylase 8 (HDAC8), which is a promising target for the development of anticancer drugs. These compounds bound into the active site of HDAC8 in a similar way to Trichostatin A (TSA), which is an HDAC8 inhibitor. This study showed that the conformation of the 2,6-DKP ring, stereochemistry, and the type of substituent on the chiral center had an important role in the binding modes. The Gibbs free energies and dissociation constants values of HDAC8-ligand complexes showed that compounds (S)-4hBn, (S)-4m, (R)-4h, and (R)-4m were more stable and affine towards HDAC8 than TSA. The inhibitory activities of 4a, (S)-4h, (S)- and (R)-4(g, l, m) were evaluated in vitro on HDAC8. It was found that compounds (R)-4g (IC50 = 21.54 nM) and (R)-4m (IC50 = 10.81 nM) exhibited better inhibitory activities than TSA (IC50 = 28.32 nM). These results suggested that 2,6-DKPs derivatives may be promising anticancer agents for further biological studies.

MIR145-3p promotes autophagy and enhances bortezomib sensitivity in multiple myeloma by targeting HDAC4.

Multiple myeloma (MM) is an incurable plasma cell malignancy with poor survival. Autophagy, a stress-responsive catabolic process mediated by lysosomal activity, plays a crucial role in the pathophysiology of MM. Growing evidence has indicated that dysregulated microRNAs (miRNAs) are associated with the aberrant autophagy in various human cancers. However, to date, few miRNAs have been reported to directly modulate autophagy in the pathobiology of MM. In this study, we investigated the role of MIR145-3p (microRNA 145-3p) in MM, with focus on cellular processes autophagy and cell death. Our results provided evidence that downregulation of MIR145-3p expression was associated with disease progression in human MM. MIR145-3p triggered autophagic flux through direct targeting of HDAC4 (histone deacetylase 4) in MM cells, leading to enhanced apoptosis. Silencing HDAC4 recapitulated the effects of MIR145-3p, whereas enforced expression of HDAC4 abrogated the effects of MIR145-3p. Furthermore, we showed that suppression of HDAC4 by MIR145-3p resulted in upregulation of the pro-apoptotic protein BCL2L11 and caused MTORC1 inactivation, which in turn led to enhanced autophagy and cell death. Importantly, we demonstrated that MIR145-3p mimic could potentiate the anti-MM activity of bortezomib in both in vitro and in vivo experiments. Overall, our findings indicate that MIR145-3p exerted a tumor suppression function in MM by inducing autophagic cell death and suggest that MIR145-3p-based targeted therapy would represent a novel strategy for MM treatment.Abbreviations: 3-MA: 3-methyladenine; 3'-UTR: 3'-untranslated region; 7-AAD: 7-aminoactinomycin D; ACTB: actin beta; ANXA5: annexin A5; ATG5: autophagy related 5; ATG7: autophagy related 7; B2M: beta-2-microglobulin; BAF: bafilomycin A1; BCL2L11: BCL2 like 11; Bort: bortezomib; CASP3: caspase 3; CCK-8: Cell Counting Kit-8; CQ: chloroquine; Ct: threshold cycle; ctrl: control; DAPI: 4',6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HDAC4: histone deacetylase 4; ISS: International Staging System; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; miRNAs: microRNAs; MIR145-3p: microRNA 145-3p; MM: multiple myeloma; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PCs: plasma cells; PFS: progression-free survival; qRT-PCR: quantitative reverse transcription PCR; RPS6KB1: ribosomal protein S6 kinase B1; SD: standard deviation; siRNA: small interfering RNA; SQSTM1: sequestosome 1; STV: starvation; TUBB: tubulin beta class I.

Anlotinib inhibits synovial sarcoma by targeting GINS1: a novel downstream target oncogene in progression of synovial sarcoma.

BACKGROUND: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. METHODS: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. RESULTS: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. CONCLUSION: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.

Chronic lead exposure decreases the expression of Huntingtin-associated protein 1 (HAP1) through Repressor element-1 silencing transcription (REST).

Chronic lead (Pb) exposure has been shown to reduce the expression of some synaptic proteins which are involved in vesicular trafficking and affect presynaptic neurotransmitter release. However, the precise mechanisms by Pb impairs neurotransmitter release are still not well defined. In the current study, we aimed to elucidate the changes of Huntingtin-associated protein 1 (HAP1) in Pb exposed rats and PC12 cells models and its molecular mechanism. Repressor element-1 silencing transcription (REST) modulates the expression of genes containing the repressor element 1 (RE-1) cis-regulatory DNA sequence. HAP1 promoter region contains a RE-1 binding motif. We also observed whether Pb exposure regulated the HAP1 transcription level through influencing the expression of REST. Mother rats were exposed to 0.5 and 2 g/L Pb acetate (PbAc) in drinking water from the first day of gestation until postnatal 21 days, then the offspring rats were continued to drink PbAc for 1 year, while the control groups received drinking water. PC12 cells were divided into 3 groups: 0 µM, 1 µM and 100 µM PbAc. The results revealed that Pb levels in blood and brain of Pb exposed groups were significantly higher than that of the control group. The ability of learning and memory in Pb exposed rats was decreased. Pb exposure reduced the expression of HAP1 and increased the REST expression. Silencing REST could reverse the decreasing of HAP1 in Pb exposed PC12 cells. Our findings raise a possibility that the decreasing of HAP1 expression by Pb exposure may affect neurotransmitter release and results in impairments in spatial learning and memory ability.

Short half-life of HPV16 E6 and E7 mRNAs sensitizes HPV16-positive tonsillar cancer cell line HN26 to DNA-damaging drugs.

Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.

PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

Immunomodulatory Nonstructural Proteins of Influenza A Viruses.

Influenza epidemics and pandemics still represent a severe public health threat and cause significant morbidity and mortality worldwide. As intracellular parasites, influenza viruses are strongly dependent on the host cell machinery. To ensure efficient production of progeny viruses, viral proteins extensively interfere with cellular signalling pathways to inhibit antiviral responses or to activate virus-supportive functions. Here, we review various functions of the influenza virus nonstructural proteins NS1, PB1-F2, and PA-X in infected cells and how post-transcriptional modifications of these proteins affect the viral life cycle. Furthermore, we discuss newly discovered interactions between these proteins and the antiviral interferon response.

MicroRNA-191, regulated by HIF-2α, is involved in EMT and acquisition of a stem cell-like phenotype in arsenite-transformed human liver epithelial cells.

Inorganic arsenic is widely distributed in the environment, and epidemiologic data show a strong association between arsenic exposure and risk of liver cancer. An understanding of the mechanisms underlying development of liver cancer and metastasis would be useful in reducing the incidence and mortality of liver cancer. MicroRNAs (miRs) act as regulators in liver cancer. Here, we show that acute or chronic exposure of human liver epithelial L-02 cells to arsenite increased expression of miR-191. There were decreased levels of BASP-1 and E-cadherin and increased levels of WT-1 and N-cadherin, indicating that arsenite induced epithelial-mesenchymal transition (EMT). Moreover, arsenite increased EpCAM and CD90 mRNA levels, showing the acquisition of stem cell-like properties by these cells. Suppression of miR-191 resulted in repression of EMT and reduced expression of stem-cell markers. Further, a miR-191 inhibitor blocked spheroid formation and production of side population cells. Luciferase reporter assays indicated that miR-191 was a target of HIF-2α, and inhibition of miR-191 decreased the neoplastic and metastatic properties of arsenite-transformed L-02 cells. Thus, in arsenite-transformed liver epithelial cells, transcriptional activation of the miR-191 promoter by HIF-2α is involved in EMT and in the acquisition of a stem cell-like phenotype.

Exercise Protects Skeletal Muscle during Chronic Doxorubicin Administration.

PURPOSE: This study aimed to assess the ability for exercise training performed before and during biweekly doxorubicin (DOX) administration to attenuate adverse effects of DOX on skeletal muscle. We hypothesized that DOX treatment would increase REDD1, impair mammalian target of rapamycin (mTOR) signaling, and reduce muscle fiber size, and that exercise training would attenuate these responses. METHODS: Eight-week-old ovariectomized female Sprague-Dawley rats were randomized to one of four treatments: exercise + DOX (Ex-Dox), Ex + vehicle (Ex-Veh), sedentary + DOX (Sed-Dox), and Sed + Veh (Sed-Veh). DOX (4 mg·kg) or vehicle (saline) intraperitoneal injections were performed biweekly for a total of three injections (cumulative dose, 12 mg·kg). Ex animals performed interval exercise (4 × 4 min, 85%-90% V˙O2peak) 5 d·wk starting 1 wk before the first injection and continued throughout study duration. Animals were euthanized ~5 d after the last injection, during which the soleus muscle was dissected and prepared for immunoblot and immunohistochemical analyses. RESULTS: REDD1 mRNA and protein were increased only in Sed-Dox (P < 0.05). The phosphorylation of mTOR and 4E-BP1 and MHC I and MHC IIa fiber size were lower in Sed-Dox versus Sed-Veh (P < 0.05). By contrast, REDD1 mRNA and protein, mTOR, 4E-BP1, and MHC I fiber size were not different between Ex-Dox and Ex-Veh (P > 0.05). LC3BI was higher, and the LC3BII/I ratio was lower in Sed-Dox versus Sed-Veh (P < 0.05) but not between Ex-Dox and Ex-Veh (P > 0.05). CONCLUSION: These data suggest that DOX may inhibit mTORC1 activity and reduce MHCI and MHCIIa fiber size, potentially through elevated REDD1, and that exercise may provide a therapeutic strategy to preserve skeletal muscle size during chronic DOX treatment.

The Novel HDAC8 Inhibitor WK2-16 Attenuates Lipopolysaccharide-Activated Matrix Metalloproteinase-9 Expression in Human Monocytic Cells and Improves Hypercytokinemia In Vivo.

Dysregulated human monocytes/macrophages can synthesize and secrete matrix metalloproteinases (MMPs), which play important roles in the progression of sepsis. In this study, we investigated the effects and mechanism of a novel histone deacetylase (HDAC8) inhibitor, (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16), on MMP-9 production and activation in stimulated human monocytic THP-1 cells. Our results demonstrated that the acetylation level of structural maintenance of chromosomes 3 (SMC3) was up-regulated by WK2-16 in THP-1 cells. Consistently, an in vitro enzyme study demonstrated that WK2-16 selectively inhibited HDAC8 activity. Moreover, the WK2-16 concentration dependently suppressed MMP-9-mediated gelatinolysis induced by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). Additionally, WK2-16 significantly inhibited both MMP-9 protein and mRNA expression without cellular toxicity. Nevertheless, WK2-16 suppressed the extracellular levels of interleukin (IL)-6 from LPS-stimulated THP-1 cells. For the signaling studies, WK2-16 had no effect on LPS/TLR4 downstream signaling pathways, such as the NF-κB and ERK/JNK/P38 MAPK pathways. On the other hand, WK2-16 enhanced the recruitment of acetylated Yin Yang 1 (YY1) with HDAC1. Finally, in vivo studies indicated that WK2-16 could reduce the serum levels of TNF-α and IL-6 in endotoxemic mice. These results suggested that HDAC8 inhibition might provide a novel therapeutic strategy of hypercytokinemia in sepsis.

The Complex Role of the ZNF224 Transcription Factor in Cancer.

ZNF224 is a member of the Kruppel-associated box zinc finger proteins (KRAB-ZFPs) family. It was originally identified as a transcriptional repressor involved in gene-specific silencing through the recruitment of the corepressor KAP1, chromatin-modifying activities, and the arginine methyltransferase PRMT5 on the promoter of its target genes. Recent findings indicate that ZNF224 can behave both as a tumor suppressor or an oncogene in different human cancers. The transcriptional regulatory properties of ZNF224 in these systems appear to be complex and influenced by specific sets of interactors. ZNF224 can also act as a transcription cofactor for other DNA-binding proteins. A role for ZNF224 in transcriptional activation has also emerged. Here, we review the state of the literature supporting both roles of ZNF224 in cancer. We also examine the functional activity of ZNF224 as a transcription factor and the influence of protein partners on its dual behavior. Increasing information on the mechanism through which ZNF224 can operate could lead to the identification of agents capable of modulating ZNF224 function, thus potentially paving the way to new therapeutic strategies for treatment of cancer.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

The anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activation.

We characterized the mechanism of action of the neuregulin-non-competitive anti-HER3 therapeutic antibody 9F7-F11 that blocks the PI3K/AKT pathway, leading to cell cycle arrest and apoptosis in vitro and regression of pancreatic and breast cancer in vivo. We found that 9F7-F11 induces rapid HER3 down-regulation. Specifically, 9F7-F11-induced HER3 ubiquitination and degradation in pancreatic, breast and prostate cancer cell lines was driven mainly by the itchy E3 ubiquitin ligase (ITCH/AIP4). Overexpression of the ITCH/AIP4 inhibitor N4BP1 or small-interfering RNA-mediated knockdown of ITCH/AIP4 inhibited HER3 ubiquitination/degradation and PI3K/AKT signaling blockade induced by 9F7-F11. Moreover, 9F7-F11-mediated JNK1/2 phosphorylation led to ITCH/AIP4 activation and recruitment to HER3 for receptor ubiquitination and degradation. ITCH/AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference. Taken together, our results suggest that 9F7-F11-induced HER3 ubiquitination and degradation in cancer cells mainly occurs through JNK1/2-dependent ITCH/AIP4 activation.