Liver epigenomic signature associated with chronic oxidative stress in a mouse model of glutathione deficiency.

Oxidative stress is intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of the ubiquitous antioxidant glutathione (GSH). Unexpectedly, chronic GSH deficiency renders glutamate-cysteine ligase modifier subunit (Gclm)-null mice protected from fatty liver injuries. Epigenetic regulation serves as an important cellular mechanism in modulating gene expression and disease outcome in FLD, although it is not well understood how systemic redox imbalance modifies the liver epigenome. In the current study, utilizing the Gclm-null mouse model, we aimed to elucidate redox-associated epigenomic changes and their implications in liver stress response. We performed high-throughput array-based DNA methylation profiling (MeDIP array) in 22,327 gene promoter regions (from -1300 bp to +500 bp of the Transcription Start Sites) in the liver and peripheral blood cells. Results from the MeDIP array demonstrate that, although global methylation enrichment in gene promoters did not change, low GSH resulted in prevalent demethylation at the individual promoter level. Such an effect likely attributed to a declined availability of the methyl donor S-adenosyl methionine (SAM) in Gclm-null liver. Functional enrichment analysis of liver target genes is suggestive of a potential role of epigenetic mechanisms in promoting cellular survival and lipid homeostasis in Gclm-null liver. In comparison with the liver tissue, MeDIP array in peripheral blood cells revealed a panel of 19 gene promoters that are candidate circulating biomarkers for hepatic epigenomic changes associated with chronic GSH deficiency. Collectively, our results provided new insights into the in vivo interplay between liver redox state and DNA methylation status. The current study laid the groundwork for future epigenetic/epigenomic investigations in experimental settings or human populations under conditions of liver oxidative stress induced by environmental or dietary challenges.

Inhibition of EZH2 exerts antitumorigenic effects in renal cell carcinoma via LATS1.

The most common type of kidney cancer in adults is renal cell carcinoma (RCC), which accounts for approximately 90% of cases. RCC is a variant disease with numerous subtypes; the most common subtype is clear cell RCC (ccRCC, 75%), followed by papillary RCC (pRCC, 10%) and chromophobe RCC (chRCC, 5%). To identify a genetic target for all subtypes, we analyzed The Cancer Genome Atlas (TCGA) databases of ccRCC, pRCC, and chromophobe RCC. Enhancer of zeste homolog 2 (EZH2), which encodes a methyltransferase, was observed to be significantly upregulated in tumors. The EZH2 inhibitor tazemetostat induced anticancer effects in RCC cells. TCGA analysis revealed that large tumor suppressor kinase 1 (LATS1), a key tumor suppressor of the Hippo pathway, was significantly downregulated in tumors; the expression of LATS1 was increased by tazemetostat. Through additional experiments, we confirmed that LATS1 plays a crucial role in EZH2 inhibition and has a negative association with EZH2. Therefore, we suggest that epigenetic control could be a novel therapeutic strategy for three subtypes of RCC.

Effect of α1D-adrenoceptor blocker for the reduction of ureteral contractions.

PURPOSE: Urolithiasis is a common urinary tract disease with growing prevalence. Alpha1-adrenoceptors (α1-ARs) are abundant in ureteral smooth muscle, distributed with different α1-AR subtypes. α1D-AR is the most widely distributed in the ureter. However, the effect of α1D-AR blockade on ureteric contraction remains unknown. MATERIALS AND METHODS: We dissected smooth muscle tissues (3 mm×3 mm) from the rat bladder and human ureter, tied silk strips on both tissue ends, and measured contraction in an organ bath chamber. Contraction activity in ureteral smooth muscle cells (USMCs) was immunocytochemically examined using primary rat and human USMC cultures. RESULTS: Using the organ bath system, we determined the inhibitory effects of silodosin, tamsulosin, and naftopidil. Naftopidil significantly decreased contractility of rat bladder tissue; similar results were observed in human ureteral tissue. To confirm ex vivo experimental results in vitro , we examined the phosphorylation of myosin light chain (MLC), a marker of contractility, in a primary human USMC culture. The examined drugs decreased phospho-MLC levels in human USMCs; however, naftopidil profoundly increased MLC dephosphorylation. CONCLUSIONS: We studied the effects of naftopidil, an α1D-AR inhibitor, on the ureter. Compared with alpha-blockers, naftopidil significantly relaxed ureteral smooth muscle. Therefore, naftopidil could be an effective therapy for patients with ureteral stones.

Therapeutic Efficacy of YM155 to Regulate an Epigenetic Enzyme in Major Subtypes of RCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and includes more than 10 subtypes. Compared to the intensively investigated clear cell RCC (ccRCC), the underlying mechanisms and treatment options of other subtypes, including papillary RCC (pRCC) and chromogenic RCC (chRCC), are limited. In this study, we analyzed the public databases for ccRCC, pRCC, and chRCC and found that BIRC5 was commonly overexpressed in a large cohort of pRCC and chRCC patients as well as ccRCC and was closely related to the progression of RCCs. We investigated the potential of BIRC5 as a therapeutic target for these three types of RCCs. Loss and gain of function studies showed the critical role of BIRC5 in cancer growth. YM155, a BIRC5 inhibitor, induced a potent tumor-suppressive effect in the three types of RCC cells and xenograft models. To determine the mechanism underlying the anti-tumor effects of YM155, we examined epigenetic modifications in the BIRC5 promoter and found that histone H3 lysine 27 acetylation (H3K27Ac) was highly enriched on the promoter region of BIRC5. Chromatin-immunoprecipitation analysis revealed that H3K27Ac enrichment was significantly decreased by YM155. Immunohistochemistry of xenografted tissue showed that overexpression of BIRC5 plays an important role in malignancy in RCC. Furthermore, high expression of P300 was significantly associated with the progression of RCC. Our findings demonstrate the P300-H3K27Ac-BIRC5 cascade in three types of RCC and provide a therapeutic path for future research on RCC.

Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma.

Sunitinib, a VEGF blockade, is used to treat clear cell renal cell carcinoma (ccRCC). However, the anti-cancer treatment effects of sunitinib do not last long in ccRCC patients. Ginsenoside, a natural medicine extracted from ginseng, has been studied in cancer treatment and shown to have anti-tumor effects and low toxicity. We assessed cell viability and cell cycle analysis in ccRCC cell lines after treatment with ginsenoside and sunitinib. DNA damage was evaluated by measuring 8-OHdG levels and comet assay. ROS levels, reflecting the cause of oxidative stress, were also measured. Ginsenoside significantly enhanced the inhibition of cell viability by sunitinib, a result that was also confirmed in the xenograft model. In cell cycle analysis, combination treatment of ginsenoside and sunitinib enhanced G2M arrest in comparison with single-treatment groups. In addition, DNA damage was increased by ginsenoside and sunitinib according to the comet assay, and the level of 8-OHdG, which reflects oxidative DNA damage, also increased. We verified that ginsenoside enhances the efficacy of sunitinib to inhibit the proliferation of ccRCC cells via induction of oxidative DNA damage. The combination therapy of sunitinib and ginsenoside suggested the possibility of effectively treating ccRCC patients.

Synergic Effect of Metformin and Everolimus on Mitochondrial Dynamics of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) frequently recurs or metastasizes after surgical resection. Everolimus, an mTOR inhibitor, is used as a second-line treatment, but the response of RCC to everolimus is insufficient. Metformin is an antidiabetic drug; recent reports have indicated its anti-cancer effects in various cancers, and it is known to have synergistic effects with other drugs. We investigated the possibility of coadministering everolimus and metformin as an effective treatment for RCC. RCC cells treated with a combination of the two drugs showed significantly inhibited cell viability, cell migration, and invasion, and increased apoptosis compared to those treated with each drug alone. An anti-cancer synergistic effect was also confirmed in the xenograft model. Transcriptome analysis for identifying the underlying mechanism of the combined treatment showed the downregulation of mitochondrial fusion genes and upregulation of mitochondrial fission genes by the combination treatment. Changes in mitochondrial dynamics following the combination treatment were observed using LysoTracker, LysoSensor, and JC-1 staining. In conclusion, the combination of everolimus and metformin inhibited RCC growth by disrupting mitochondrial dynamics. Therefore, we suggest that a treatment combining metformin and everolimus disrupts mitochondrial dynamics in RCC, and may be a novel strategy for RCC treatment.

Entinostat, a histone deacetylase inhibitor, increases the population of IL-10+ regulatory B cells to suppress contact hypersensitivity.

IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].

Indwelling urinary catheter assembled with lidocaine-loaded polymeric strand for local sustained alleviation of bladder discomfort.

Indwelling urinary catheters (IUCs) are used in clinical settings to assist detrusor contraction in hospitalized patients. However, an inserted IUC often causes catheter-related bladder discomfort. To resolve this, we propose an IUC coupled with local, sustained release of an anesthetic drug, lidocaine. For this, a thin strand composed of poly (lactic-co-glycolic acid) and lidocaine was separately prepared as a drug delivery carrier, which was then wound around the surface of the IUC to produce the drug-delivery IUC. Our results revealed that the drug-delivery IUC could exert the pain-relief effects for up to 7 days when placed in the bladder of living rats. Cystometrogram tests indicated that the drug-delivery IUC could significantly relieve bladder discomfort compared with the IUC without lidocaine. Furthermore, the expression of pain-related inflammatory markers, such as nerve growth factor, cyclooxygenase-2, and interleukin-6 in the biopsied bladder tissues was significantly lower when the drug-delivery IUC was used. Therefore, we conclude that an IUC simply assembled with a drug-loaded polymer strand can continuously release lidocaine to allow for the relief of bladder discomfort during the period of IUC insertion.

Nucleoporin 210 Serves a Key Scaffold for SMARCB1 in Liver Cancer.

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. SIGNIFICANCE: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

Catheter-Related Bladder Discomfort: How Can We Manage It?

The urethral catheter is used in various clinical situations such as diagnosing urologic disease, urine drainage in patients after surgery, and for patients who cannot urinate voluntarily. However, catheters can cause numerous adverse effects, such as catheter-associated infection, obstruction, bladder stones, urethral injury, and catheter-related bladder discomfort (CRBD). CRBD symptoms vary among patients from burning sensation and pain in the suprapubic and penile areas to urinary urgency. CRBD significantly reduces patient quality of life and can lead to several complications. CRBD is caused by catheter-induced bladder irritation due to muscarinic receptor-mediated involuntary contractions of bladder smooth muscle and also can be caused by mechanical stimulus of the urethral catheter. Various pharmacologic studies for managing CRBD, including antimuscarinic and antiepileptic agents and botulinum toxin injections have been reported. If urologists can reduce patients' CRBD, their quality of life and recovery can improve.

Drug repositioning of TANK-binding kinase 1 inhibitor CYT387 as an alternative for the treatment of Gram-negative bacterial sepsis.

There is currently no specific drug for the treatment of sepsis and antibiotic administration is considered the best option, despite numerous issues. Therefore, the development of drugs to control the pathogen-induced inflammatory responses associated with sepsis is essential. To address this, our study examined the transcriptomes of lipopolysaccharide (LPS)-induced dendritic cells (DCs), identifying TANK-binding kinase1 (Tbk1) as a key factor involved in the inflammatory response. These data suggested drug repositioning of the Tbk1 inhibitor CYT387, currently used for the treatment of myelofibrosis and some cancers, as a candidate for regulating the LPS-induced inflammatory response. CYT387 also inhibited pro-inflammatory cytokine and surface molecule expression by mature DCs after LPS exposure. These effects correlated with both Akt phosphorylation and IκBα degradation. Finally, CYT387 demonstrated therapeutic effects in LPS-induced endotoxemia and Escherichia coli K1-induced mouse models of sepsis and decreased the expression of pro-inflammatory cytokines. In conclusion, our study suggests that drug repositioning of CYT387 may serve as a potential therapeutic for sepsis.

SOX9 is controlled by the BRD4 inhibitor JQ1 via multiple regulation mechanisms.

SOX9 is a key transcription factor during cell differentiation, sex determination, and tumorigenesis. However, the detailed mechanisms of its targeting strategy remain elusive. To investigate possibilities of targeting SOX9 with epigenetic drugs and the precise underlying mechanisms, two human cancer cell lines were chosen as model systems, which showed high SOX9 expression and anti-tumorigenic effects upon loss of SOX9. Histone acetylation-related screening of a small panel of epigenetic drugs revealed that the bromodomain reader inhibitor JQ1 dramatically downregulated SOX9 through multiple regulation steps, namely, transcription, BRD4-SOX9 protein-protein interaction, and further protein stability. These findings suggest that BRD4 inhibition is a novel therapeutic strategy for diseases characterized by SOX9 overexpression.

Oncogenic IL7R is downregulated by histone deacetylase inhibitor in esophageal squamous cell carcinoma via modulation of acetylated FOXO1.

The interleukin-7 receptor (IL7R) is generally expressed in immune cells and is critical in survival, development and homeostasis in the immune system. Advanced genome-wide cancer studies have reported that IL7R is genetically amplified in human esophageal squamous cell carcinoma (ESCC), however, the exact role of IL7R in ESCC has not been investigated. In the present study, it was found that IL7R was overexpressed in ESCC cohorts and the loss of IL7R induced anti-oncogenic effects in ESCC cell lines. A small panel of epigenetic drugs were screened for their ability to downregulate the expression of IL7R. Unexpectedly, apicidin, a histone deacetylase (HDAC) inhibitor, effectively downregulated the expression of IL7R in a dose-dependent manner at an early time-point, as determined by quantitative polymerase chain reaction and IL7R immunostaining, and did not require de novo protein synthesis. Of note, apicidin induced the acetylation of Forkhead box-containing protein, O subfamily 1, which acts as a repressor at the IL7R promoter, accompanied with depleted active histone modifications based on chromatin immunoprecipitation assay. Taken together, these results demonstrated that targeting oncogenic IL7R in ESCC by HDAC inhibitors may be a valuable therapeutic approach.

Epigenetic landscape change analysis during human EMT sheds light on a key EMT mediator TRIM29.

Epithelial to mesenchymal transition (EMT) is a key trans-differentiation process, which plays a critical role in physiology and pathology. Although gene expression changes in EMT have been scrutinized, study of epigenome is in its infancy. To understand epigenetic changes during TWIST-driven EMT, we used the AcceSssIble assay to study DNA methylation and chromatin accessibility in human mammary epithelial cells (HMECs). The DNA methylation changes were found to have functional significance in EMT - i.e. methylated genes were enriched for E-box motifs that can be recognized by TWIST, at the promoters suggesting a potential targeting phenomenon, whereas the demethylated regions were enriched for pro-metastatic genes, supporting the role of EMT in metastasis. TWIST-induced EMT triggers alterations in chromatin accessibility both independent of and dependent on DNA methylation changes, primarily resulting in closed chromatin conformation. By overlapping the genes, whose chromatin structure is changed during early EMT and a known "core EMT signature", we identified 18 driver candidate genes during EMT, 14 upregulated and 4 downregulated genes with corresponding chromatin structure changes. Among 18 genes, we focused on TRIM29 as a novel marker of EMT. Although loss of TRIM29 is insufficient to suppress CDH, it is enough to induce CDH2 and VIM. Gene functional annotation analysis shows the involvement of TRIM29 in epidermal development, cell differentiation and cell migration. Taken together, our results provide a robust snapshot of chromatin state during human EMT and identify TRIM29 as a core mediator of EMT.

JQ1, a BET inhibitor, controls TLR4-induced IL-10 production in regulatory B cells by BRD4-NF-κB axis.

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomaincontaining protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer. [BMB Reports 2017; 50(12): 640-646].

Epigenetic reader BRD4 inhibition as a therapeutic strategy to suppress E2F2-cell cycle regulation circuit in liver cancer.

Deregulation of the epigenome component affects multiple pathways in the cancer phenotype since the epigenome acts at the pinnacle of the hierarchy of gene expression. Pioneering work over the past decades has highlighted that targeting enzymes or proteins involved in the epigenetic regulation is a valuable approach to cancer therapy. Very recent results demonstrated that inhibiting the epigenetic reader BRD4 has notable efficacy in diverse cancer types. We investigated the potential of BRD4 as a therapeutic target in liver malignancy. BRD4 was overexpressed in three different large cohort of hepatocellular carcinoma (HCC) patients as well as in liver cancer cell lines. BRD4 inhibition by JQ1 induced anti-tumorigenic effects including cell cycle arrest, cellular senescence, reduced wound healing capacity and soft agar colony formation in liver cancer cell lines. Notably, BRD4 inhibition caused MYC-independent large-scale gene expression changes in liver cancer cells. Serial gene expression analyses with SK-Hep1 liver cancer cells treated with JQ1 to delineate the key player of BRD4 inhibition identified E2F2 as the first line of downstream direct target of BRD4. Further experiments including chromatin immunoprecipitation (ChIP) assay and loss of function study confirmed E2F2 as key player of BRD4 inhibition. Overexpressed E2F2 is a crucial center of cell cycle regulation and high expression of E2F2 is significantly associated with poor prognosis of HCC patients. Our findings reveal BRD4-E2F2-cell cycle regulation as a novel molecular circuit in liver cancer and provide a therapeutic strategy and innovative insights for liver cancer therapies.

JQ1, an inhibitor of the epigenetic reader BRD4, suppresses the bidirectional MYC-AP4 axis via multiple mechanisms.

Bromodomain and extra-terminal domain (BET) family proteins are representative epigenetic modulators that read acetylated lysine residues and transfer cellular signals. Recently, the BET protein inhibitor JQ1 was developed and has been extensively studied in many cancer cell types. We demonstrated that JQ1 effectively suppressed the MYC-AP4 axis and induced antitumorigenic effects by targeting a bidirectional positive loop between MYC and AP4 which was first proposed in the present study. MYC and AP4 are the direct targets of BRD4, as demonstrated by chromatin immunoprecipitation (ChIP) assay and BRD4 loss-of-function experiments. Although inhibition of the MYC/MAC dimer suppressed AP4, the efficacy of suppression was not as effective as BRD4 inhibition. Notably, AP4 loss-of-function studies demonstrated that AP4 is a major critical target of JQ1 and that MYC is a novel downstream target of AP4, as demonstrated by AP4 binding to the MYC promoter. Taken together, our results suggest that the epigenetic reader BRD4 is a key mediator of the activated MYC-AP4 axis, which supports the possibility that targeting BET protein is a novel therapeutic strategy for MYC-AP4 axis-activated cancers.

The Epigenetic Reader BRD2 as a Specific Modulator of PAI-1 Expression in Lipopolysaccharide-Stimulated Mouse Primary Astrocytes.

The post translational modification of lysine acetylation is a key mechanism that regulates chromatin structure. Epigenetic readers, such as the BET domains, are responsible for reading histone lysine acetylation which is a hallmark of open chromatin structure, further providing a scaffold that can be accessed by RNA polymerases as well as transcription factors. Recently, several reports have assessed and highlighted the roles of epigenetic readers in various cellular contexts. However, little is known about their role in the regulation of inflammatory genes, which is critical in exquisitely tuning inflammatory responses to a variety of immune stimuli. In this study, we investigated the role of epigenetic readers BRD2 and BRD4 in the lipopolysaccharide (LPS)-induced immune responses in mouse primary astrocytes. Inflammatory stimulation by LPS showed that the levels of Brd2 mRNA and protein were increased, while Brd4 mRNA levels did not change. Knocking down of Brd2 mRNA using specific small interfering RNA (siRNA) in cultured mouse primary astrocytes inhibited LPS-induced mRNA expression and secretion of plasminogen activator inhibitor-1 (PAI-1). However, no other pro-inflammatory cytokines, such as Il-6, Il-1ß and Tnf-α, were affected. Indeed, treatment with bromodomain-containing protein inhibitor, JQ1, blocked Pai-1 mRNA expression through the inhibition of direct BRD2 protein-binding and active histone modification on Pai-1 promoter. Taken together, our data suggest that BRD2 is involved in the modulation of neuroinflammatory responses through PAI-1 and via the regulation of epigenetic reader BET protein, further providing a potential novel therapeutic strategy in neuroinflammatory diseases.