The RNA polymerase II subunit B (RPB2) functions as a growth regulator in human glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor with a poor prognosis. The growth of GBM cells depends on the core transcriptional apparatus, thus rendering RNA polymerase (RNA pol) complex as a candidate therapeutic target. The RNA pol II subunit B (POLR2B) gene encodes the second largest subunit of the RNA pol II (RPB2); however, its genomic status and function in GBM remain unclear. Certain GBM data sets in cBioPortal were used for investigating the genomic status and expression of POLR2B in GBM. The function of RPB2 was analyzed following knockdown of POLR2B expression by shRNA in GBM cells. The cell counting kit-8 assay and PI staining were used for cell proliferation and cell cycle analysis. A xenograft mouse model was established to analyze the function of RPB2 in vivo. RNA sequencing was performed to analyze the RPB2-regulated genes. GO and GSEA analyses were applied to investigate the RPB2-regulated gene function and associated pathways. In the present study, the genomic alteration and overexpression of the POLR2B gene was described in glioblastoma. The data indicated that knockdown of POLR2B expression suppressed tumor cell growth of glioblastoma in vitro and in vivo. The analysis further demonstrated the identification of the RPB2-regulated gene sets and highlighted the DNA damage-inducible transcript 4 gene as the downstream target of the POLR2B gene. The present study provides evidence indicating that RPB2 functions as a growth regulator in glioblastoma and could be used as a potential therapeutic target for the treatment of this disease.

The BRD4 Inhibitor dBET57 Exerts Anticancer Effects by Targeting Superenhancer-Related Genes in Neuroblastoma.

Neuroblastoma (NB) is the most common solid tumor of the neural crest cell origin in children and has a poor prognosis in high-risk patients. The oncogene MYCN was found to be amplified at extremely high levels in approximately 20% of neuroblastoma cases. In recent years, research on the targeted hydrolysis of BRD4 to indirectly inhibit the transcription of the MYCN created by proteolysis targeting chimaera (PROTAC) technology has become very popular. dBET57 (S0137, Selleck, TX, USA) is a novel and potent heterobifunctional small molecule degrader based on PROTAC technology. The purpose of this study was to investigate the therapeutic effect of dBET57 in NB and its potential mechanism. In this study, we found that dBET57 can target BRD4 ubiquitination and disrupt the proliferation ability of NB cells. At the same time, dBET57 can also induce apoptosis, cell cycle arrest, and decrease migration. Furthermore, dBET57 also has a strong antiproliferation function in xenograft tumor models in vivo. In terms of mechanism, dBET57 targets the BET protein family and the MYCN protein family by associating with CRBN and destroys the SE landscape of NB cells. Combined with RNA-seq and ChIP-seq public database analysis, we identified the superenhancer-related genes TBX3 and ZMYND8 in NB as potential downstream targets of dBET57 and experimentally verified that they play an important role in the occurrence and development of NB. In conclusion, these results suggest that dBET57 may be an effective new therapeutic drug for the treatment of NB.

Super-enhancer profiling identifies novel critical and targetable cancer survival gene LYL1 in pediatric acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a myeloid neoplasm makes up 7.6% of hematopoietic malignancies. Super-enhancers (SEs) represent a special group of enhancers, which have been reported in multiple cell types. In this study, we explored super-enhancer profiling through ChIP-Seq analysis of AML samples and AML cell lines, followed by functional analysis. METHODS: ChIP-seq analysis for H3K27ac was performed in 11 AML samples, 7 T-ALL samples, 8 B-ALL samples, and in NB4 cell line. Genes and pathways affected by GNE-987 treatment were identified by gene expression analysis using RNA-seq. One of the genes associated with super-enhancer and affected by GNE-987 treatment was LYL1 basic helix-loop-helix family member (LYL1). shRNA mediated gene interference was used to down-regulate the expression of LYL1 in AML cell lines, and knockdown efficiency was detected by RT-qPCR and western blotting. The effect of knockdown on the growth of AML cell lines was evaluated by CCK-8. Western blotting was used to detect PARP cleavage, and flow cytometry were used to determine the effect of knockdown on apoptosis of AML cells. RESULTS: We identified a total of 200 genes which were commonly associated with super-enhancers in ≧10 AML samples, and were found enriched in regulation of transcription. Using the BRD4 inhibitor GNE-987, we assessed the dependence of AML cells on transcriptional activation for growth and found GNE-987 treatment predominantly inhibits cell growth in AML cells. Moreover, 20 candidate genes were selected by super-enhancer profile and gene expression profile and among which LYL1 was observed to promote cell growth and survival in human AML cells. CONCLUSIONS: In summary, we identified 200 common super-enhancer-associated genes in AML samples, and a series of those genes are cancer genes. We also found GNE-987 treatment downregulates the expression of super-enhancer-associated genes in AML cells, including the expression of LYL1. Further functional analysis indicated that LYL1 is required for AML cell growth and survival. These findings promote understanding of AML pathophysiology and elucidated an important role of LYL1 in AML progression.

BRD4 inhibitor GNE987 exerts anti-cancer effects by targeting super-enhancers in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a common extracranial malignancy with high mortality in children. Recently, super-enhancers (SEs) have been reported to play a critical role in the tumorigenesis and development of NB via regulating a wide range of oncogenes Thus, the synthesis and identification of chemical inhibitors specifically targeting SEs are of great urgency for the clinical therapy of NB. This study aimed to characterize the activity of the SEs inhibitor GNE987, which targets BRD4, in NB. RESULTS: In this study, we found that nanomolar concentrations of GNE987 markedly diminished NB cell proliferation and survival via degrading BRD4. Meanwhile, GNE987 significantly induced NB cell apoptosis and cell cycle arrest. Consistent with in vitro results, GNE987 administration (0.25 mg/kg) markedly decreased the tumor size in the xenograft model, with less toxicity, and induced similar BRD4 protein degradation to that observed in vitro. Mechanically, GNE987 led to significant downregulation of hallmark genes associated with MYC and the global disruption of the SEs landscape in NB cells. Moreover, a novel candidate oncogenic transcript, FAM163A, was identified through analysis of the RNA-seq and ChIP-seq data. FAM163A is abnormally transcribed by SEs, playing an important role in NB occurrence and development. CONCLUSION: GNE987 destroyed the abnormal transcriptional regulation of oncogenes in NB by downregulating BRD4, which could be a potential therapeutic candidate for NB.

SAPCD2 promotes neuroblastoma progression by altering the subcellular distribution of E2F7.

Recent studies uncovered the emerging roles of SAPCD2 (suppressor anaphase-promoting complex domain containing 2) in several types of human cancer. However, the functions and underlying mechanisms of SAPCD2 in the progression of neuroblastoma (NB) remain elusive. Herein, through integrative analysis of public datasets and regulatory network of GSK-J4, a small-molecule drug with anti-NB activity, we identified SAPCD2 as an appealing target with a high connection to poor prognosis in NB. SAPCD2 promoted NB progression in vitro and in vivo. Mechanistically, SAPCD2 could directly bind to cytoplasmic E2F7 but not E2F1, alter the subcellular distribution of E2F7 and regulate E2F activity. Among the E2F family members, the roles of E2F7 in NB are poorly understood. We found that an increasing level of nuclear E2F7 was induced by SAPCD2 knockdown, thereby affecting the expression of genes involved in the cell cycle and chromosome instability. In addition, Selinexor (KTP-330), a clinically available inhibitor of exportin 1 (XPO1), could induce nuclear accumulation of E2F7 and suppress the growth of NB. Overall, our studies suggested a previously unrecognized role of SAPCD2 in the E2F signaling pathway and a potential therapeutic approach for NB, as well as clues for understanding the differences in subcellular distribution of E2F1 and E2F7 during their nucleocytoplasmic shuttling.

CEBPG promotes acute myeloid leukemia progression by enhancing EIF4EBP1.

BACKGROUND: Acute myeloid leukemia (AML) is a myeloid neoplasm accounts for 7.6% of hematopoietic malignancies. AML is a complex disease, and understanding its pathophysiology is contributing to the improvement in the treatment and prognosis of AML. In this study, we assessed the expression profile and molecular functions of CCAAT enhancer binding protein gamma (CEBPG), a gene implicated in myeloid differentiation and AML progression. METHODS: shRNA mediated gene interference was used to down-regulate the expression of CEBPG in AML cell lines, and knockdown efficiency was detected by RT-qPCR and western blotting. The effect of knockdown on the growth of AML cell lines was evaluated by CCK-8. Western blotting was used to detect PARP cleavage, and flow cytometry were used to determine the effect of knockdown on apoptosis of AML cells. Genes and pathways affected by knockdown of CEBPG were identified by gene expression analysis using RNA-seq. One of the genes affected by knockdown of CEBPG was Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), a known repressor of translation. Knockdown of EIF4EBP1 was used to assess its potential role in AML progression downstream of CEBPG. RESULTS: We explored the ChIP-Seq data of AML cell lines and non-AML hematopoietic cells, and found CEBPG was activated through its distal enhancer in AML cell lines. Using the public transcriptomic dataset, the Cancer Cell Line Encyclopedia (CCLE) and western blotting, we also found CEBPG was overexpressed in AML. Moreover, we observed that CEBPG promotes AML cell proliferation by activating EIF4EBP1, thus contributing to the progression of AML. These findings indicate that CEBPG could act as a potential therapeutic target for AML patients. CONCLUSION: In summary, we systematically explored the molecular characteristics of CEBPG in AML and identified CEBPG as a potential therapeutic target for AML patients. Our findings provide novel insights into the pathophysiology of AML and indicate a key role for CEBPG in promoting AML progression.

MI-773, a breaker of the MDM2/p53 axis, exhibits anticancer effects in neuroblastoma via downregulation of INSM1.

Neuroblastoma (NB) is a common pediatric malignancy associated with poor outcomes. Recent studies have shown that murine double minute2 homolog (MDM2) protein inhibitors are promising anticancer agents. MI-773 is a novel and specific antagonist of MDM2, however, the molecular mechanism of its anti-NB activity remains unclear. NB cell viability was measured by Cell Counting Kit-8 assay following MI-773 treatment. Cell cycle progression was analyzed using PI staining and apoptosis was assessed using Annexin V/PI staining. The molecular mechanisms by which MI-773 exerted its effects were investigated using a microarray. The results showed that disturbance of the MDM2/p53 axis by MI-773 resulted in potent suppression of proliferation, induction of apoptosis and cell cycle arrest in NB cells. In addition, microarray analysis showed that MI-773 led to significant downregulation of genes involved in the G2/M phase checkpoint and upregulation of hallmark gene associated with the p53 pathway. Meanwhile, knockdown of insulinoma-associated 1 decreased proliferation and increased apoptosis of NB cells. In conclusion, the present study demonstrated that MI-773 exhibited high selectivity and blockade affinity for the interaction between MDM2 and TP53 and may serve as a novel strategy for the treatment of NB.

COCH predicts survival and adjuvant TACE response in patients with HCC.

The aim of the present study was to measure the expression of Cochlin (COCH) and analyze its association with survival, recurrence and the benefits from adjuvant transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) following hepatectomy. Patients with high COCH expression levels had a poorer prognosis in terms of overall and disease-free survival rate compared with those with low COCH expression levels. Further analysis revealed that patients with low COCH expression who received TACE experienced markedly lower early recurrence rates compared with those who did not receive TACE. However, patients with high COCH expression with and without adjuvant TACE after resection experienced no difference in disease recurrence rates. The expression of COCH was found to be associated with hepatitis B virus infection, portal vein tumor thrombosis and Barcelona Clinic Liver Cancer stage in HCC. Therefore, the findings of the present study indicated that clinical detection of COCH expression may help estimate the prognosis of patients with HCC, as well as determine whether to administer TACE after surgery to prevent recurrence.

Interpretation of the development of neoadjuvant therapy for gastric cancer based on the vicissitudes of the NCCN guidelines.

Gastric cancer is one of the most common digestive system tumors in China, and locally advanced gastric cancer (LAGC) accounts for a high proportion of newly diagnosed cases. Although surgery is the main treatment for gastric cancer, surgical excision alone cannot achieve satisfactory outcomes in LAGC patients. Neoadjuvant therapy (NAT) has gradually become the standard treatment for patients with LAGC, and this treatment can not only achieve tumor downstaging and improve surgical rate and the R0 resection rate, but it also significantly improves the long-term prognosis of patients. Peri/preoperative neoadjuvant chemotherapy and preoperative chemoradiotherapy are both recommended according to a large number of studies, and the regimens have also been evolved in the past decades. Since the NCCN guidelines for gastric cancer are one of the most authoritative evidence-based guidelines worldwide, here, we demonstrate the development course and major breakthroughs of NAT for gastric cancer based on the vicissitudes of the NCCN guidelines from 2007 to 2019, and also discuss the future of NAT.

[Predictive Factors of Early Weight Loss after Laparoscopic Sleeve Gastrectomy].

Objective To explore the clinical factors that can be used to predict the early weight loss after laparoscopic sleeve gastrectomy(LSG).Methods The clinical data of 64 obese patients undergoing LSG in Peking Union Medical College Hospital from August 2015 to January 2018 were retrospectively analyzed.We analyzed the relationship between different clinical factors and early weight loss,determined the independent predictors based on Logistic models,and estimated their test power by using the receiver operating characteristic(ROC)curves.Results Correlation analysis indicated that preoperative body mass index(P=0.000,P=0.000,P=0.000),waist circumference(WC)(P=0.000,P=0.000,P=0.000),whole body fat volume(P=0.000,P=0.006,P=0.003),homeostatic model assessment for insulin resistance(HOMA-IR)(P=0.000,P=0.000,P=0.002),and hypersensitive C-reactive protein(hsCRP)(P=0.004,P=0.002,P=0.025)were negatively correlated with excess weight loss percentage(EWL %) after 3,6 and 12 months.Also,hsCRP after 6 months showed negative correlation with EWL % after 1 year(P=0.029).Binary Logistic regression analysis showed that WC was an independent predictor of early weight loss(P=0.018).ROC analysis showed that when the optimal cutoff value is 142.5 cm for WC,Youden index was highest,with a sensitivity of 80% and a specificity of 87%.Patients were further divided into low WC group and high WC group based on this optimal cutoff value.The low WC group had significantly higher EWL% than the high WC group 3 months(t=6.677,P=0.000),6 months(t=6.157,P=0.000),and 1 year(t=4.006,P=0.000)after surgery.The low WC group also had significantly lower hsCRP than high WC group 6 months after surgery(z=-3.510,P=0.000).HOMA-IR showed no significant difference between these two groups(z=-0.821,P=0.412).Conclusions WC is an independent predictor of weight loss early after LSG.The patients with low WC have better weight loss effectiveness.

Transcriptome and Metabolome Analyses Provide Insights into the Occurrence of Peel Roughing Disorder on Satsuma Mandarin (Citrus unshiu Marc.) Fruit.

Roughing disorder (RD) is a significant quality barrier in citrus fruit, prevalent on easy-peeling mandarins. As RD is not yet well-understood, this study aimed to examine the changes and synergic molecular processes involved in peel RD. Peel with RD was induced by severely defruiting Satsuma mandarin trees. Morphology observations, RNA-sequencing, and targeted and untargeted metabolic analyses were conducted. The results showed that the primary metabolites of sugars, organic acids and amino acids are dramatically changed in RD peel. The RD peel was always characterized by higher magnesium content during development. Comparative transcriptome profiling was performed for CK and RD peels at 30, 80, and 170 days after full bloom (DAFB) which represented fruit at cell division stage, cell enlargement stage and fruit maturity stage, respectively. Physiological and molecular biological evidence suggested that the month after full bloom is a crucial stage for RD initiation. A total of 4,855 differentially expressed genes (DEGs) in RD peel, relative to CK peel were detected at cell division stage, about 2 to 4-fold more than other stages had. Among the differentially expressed transcription factors, the bHLH family were affected most by RD, and six bHLH transcription factors functionally involved in GA metabolism were assessed to associate with RD occurrence. Gene set enrichment analysis suggested that RD significantly altered starch and GA metabolism in peel. Higher starch content and hydrolysed chain status were found in RD peel at cell division stage. RD occurrence on the peel was influenced significantly by GA, especially abundant GA before July. These changes may mean a significant alteration in sink strength of RD peel. The findings of this study provide insights into the emergence, development and molecular mechanisms of RD.