BRD9 inhibition promotes PUMA-dependent apoptosis and augments the effect of imatinib in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are primarily characterized by activating mutations of tyrosine kinase or platelet-derived growth factor receptor alpha. Although the revolutionary therapeutic outcomes of imatinib are well known, the long-term benefits of imatinib are still unclear. The effects of BRD9, a recently identified subunit of noncanonical BAF complex (ncBAF) chromatin remodeling complexes, in GISTs are not clear. In the current study, we evaluated the functional role of BRD9 in GIST progression. Our findings demonstrated that the expression of BRD9 was upregulated in GIST tissues. The downregulation or inhibition of BRD9 could significantly reduce cellular proliferation, and facilitates apoptosis in GISTs. BRD9 inhibition could promote PUMA-dependent apoptosis in GISTs and enhance imatinib activity in vitro and in vivo. BRD9 inhibition synergizes with imatinib in GISTs by inducing PUMA upregulation. Mechanism study revealed that BRD9 inhibition promotes PUMA induction via the TUFT1/AKT/GSK-3β/p65 axis. Furthermore, imatinib also upregulates PUMA by targeting AKT/GSK-3β/p65 axis. In conclusion, our results indicated that BRD9 plays a key role in the progression of GISTs. Inhibition of BRD9 is a novel therapeutic strategy in GISTs treated alone or in combination with imatinib.Subject terms: Chemotherapy, Gastric cancer     

GSTT1 Copy Number Gain and ZNF Overexpression Are Predictors of Poor Response to Imatinib in Gastrointestinal Stromal Tumors

Oncogenic mutations in gastrointestinal stromal tumors (GISTs) predict prognosis and therapeutic responses to imatinib. In wild-type GISTs, the tumor-initiating events are still unknown, and wild-type GISTs are resistant to imatinib therapy. We performed an association study between copy number alterations (CNAs) identified from array CGH and gene expression analyses results for four wild-type GISTs and an imatinib-resistant PDGFRA D842V mutant GIST, and compared the results to those obtained from 27 GISTs with KIT mutations. All wild-type GISTs had multiple CNAs, and CNAs in 1p and 22q that harbor the SDHB and GSTT1 genes, respectively, correlated well with expression levels of these genes. mRNA expression levels of all SDH gene subunits were significantly lower (P≤0.041), whereas mRNA expression levels of VEGF (P=0.025), IGF1R (P=0.026), and ZNFs (P<0.05) were significantly higher in GISTs with wild-type/PDGFRA D842V mutations than GISTs with KIT mutations. qRT-PCR validation of the GSTT1 results in this cohort and 11 additional malignant GISTs showed a significant increase in the frequency of GSTT1 CN gain and increased mRNA expression of GSTT1 in wild-type/PDGFRA D842V GISTs than KIT-mutant GISTs (P=0.033). Surprisingly, all four malignant GISTs with KIT exon 11 deletion mutations with primary resistance to imatinib had an increased GSTT1 CN and mRNA expression level of GSTT1. Increased mRNA expression of GSTT1 and ZNF could be predictors of a poor response to imatinib. Our integrative approach reveals that for patients with wild-type (or imatinib-resistant) GISTs, attempts to target VEGFRs and IGF1R may be reasonable options.     

Gastrointestinal stromal tumors: key to diagnosis and choice of therapy

The common feature of gastrointestinal stromal tumors (GISTs) is the expression of KIT protein or acquisition of activating, constitutive mutations in the KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes that are the early oncogenic events during GIST development. With these discoveries, GIST has emerged as a distinct sarcoma entity, enabling the introduction of targeted therapy using the inhibition of KIT/PDGFRA and their downstream signaling cascade. The introduction of a small-molecule tyrosine kinase inhibitor, imatinib mesylate, to clinical practice has revolutionized the treatment of patients with advanced GISTs and is currently approved as first-line treatment for patients with metastatic and/or inoperable GISTs. Mutation screening is currently a tool in GIST diagnosis, assessment of sensitivity to tyrosine kinase inhibitors, and prediction of achieving response to molecularly targeted therapy.This article discusses the histologic and molecular criteria for distinguishing GISTs from other types of sarcoma, and the molecular diagnostic tools that are currently available or in development to assist in therapy decisions.     

Practical Role of Mutation Analysis for Imatinib Treatment in Patients With Advanced Gastrointestinal Stromal Tumors: A Meta-Analysis

Background

Imatinib has become the standard first line treatment of gastrointestinal stromal tumors (GIST) in the advanced phase and adjuvant setting. We carried out an up-to-date meta-analysis to determine the practical role of mutation analysis for imatinib treatment in patients with advanced GIST.

Methods

Eligible studies were limited to imatinib treatment for patients with advanced GIST and reported on mutation analysis. Statistical analyses were conducted to calculate the odds ratio (OR), hazard ratio (HR) and 95% confidence interval (CI) using fixed-effects and random-effects models.

Results

A total of 2834 patients from 3 randomized controlled trials and 12 cohort studies were included. The ORs of response rates in KIT exon 11-mutant GISTs were 3.504 (95% CI 2.549-4.816, p<0.001) and 3.521 (95% CI 1.731-7.165, p=0.001) compared with KIT exon 9-mutant and wild type GISTs, respectively. The HRs of progression-free survival in KIT exon 11-mutant GISTs were 0.365 (95% CI 0.301-0.444, p<0.001) and 0.375 (95% CI 0.270-0.519, p<0.001) compared with KIT exon 9-mutant and wild type GISTs. The HRs of overall survival in KIT exon 11-mutant GISTs were 0.388 (95% CI 0.293-0.515, p<0.001) and 0.400 (95% CI 0.297-0.538, p<0.001) compared with KIT exon 9-mutant and wild type GISTs. No statistical significant differences were found between KIT exon 9-mutant and wild type. The overall response rate in KIT-exon 11-mutant GISTs were 70.5% (65%-75.9%) compared with 57.1% (51%-63.2%) in KIT-positive GISTs. No evidence of publication bias was observed.

Conclusion

Patients with advanced GIST harboring a KIT exon 11 mutation have the best response rate and long-term survival with imatinib treatment. Mutation analysis would be more helpful than KIT expression analysis to decide appropriate therapy for a specific patient.     

Prognostic Indicators for Gastrointestinal Stromal Tumors: A Review

Gastrointestinal stromal tumors (GISTs) are potentially malignancies that can occur anywhere in the digestive tract. Tyrosine kinase inhibitors (TKIs) such as imatinib have proven effective since the discovery of KIT and PDGFRA. The current version of NCNN, ESMO and EURACAN guidelines recognized that the three main prognostic factors are the mitotic rate, tumor size and tumor site. In addition, tumor rupture is also recognized as an independent risk factor. However, recent evidence shows that various types of gene mutations are associated with prognosis, and influencing factors such as gastrointestinal bleeding and high Ki67 index have been associated with poor prognosis. It shows that the current risk classification is still insufficient and controversial. With the emergence of more and more lack mutation in KIT/PDGFRA GISTs (KIT/PDGFRA wild-type GISTs) or drug resistance genes, primary and secondary drug resistance problems are caused, which makes the treatment of late or metastatic GIST face challenges. Therefore, this article will review the clinicopathological characteristics of GIST, the special molecular subtypes and other factors that may affect prognosis. We will also explore reliable prognostic markers for better postoperative management and improve the prognosis of patients with GIST.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Inhibition of autophagy enhances apoptosis induced by the PI3K/AKT/mTor inhibitor NVP‐BEZ235 in renal cell carcinoma cells

The PI3K/AKT/mTOR pathway plays a key role in the development of the hypervascular tumor renal cell carcinoma (RCC). NVP‐BEZ235 (NVP), a novel dual PI3K/mTOR inhibitor, showed great antitumor benefit and provided a treatment strategy in RCC. In this study, we test the effect of NVP on survival rate, apoptosis and autophagy in the RCC cell line, 786‐0. We also explore the hypothesis that NVP, in combination with autophagy inhibitors, leads to apoptosis enhancement in 786‐0 cells. The results showed that the PI3K/AKT/mTOR pathway proteins p‐AKT and p‐P70S6K were highly expressed in RCC tissue. We also showed that NVP inhibited cell growth and induced apoptosis and autophagy in RCC cells. The combination treatment of NVP with autophagy inhibitors enhanced the effect of NVP on suppressing 786‐0 growth and induction of apoptosis. This study proposes a novel treatment paradigm where combining PI3K/AKT/mTOR pathway inhibitors and autophagy inhibitors lead to enhanced RCC cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.     

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N‐RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP‐BEZ235. The two‐drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl‐2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p‐ERK, p‐AKT, p‐S6, and 4E‐BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Antitumor Effect of the Tyrosine Kinase Inhibitor Nilotinib on Gastrointestinal Stromal Tumor (GIST) and Imatinib-Resistant GIST Cells

Despite the benefits of imatinib for treating gastrointestinal stromal tumors (GIST), the prognosis for high risk GIST and imatinib-resistant (IR) GIST remains poor. The mechanisms of imatinib resistance have not yet been fully clarified. The aim of the study was to establish imatinib-resistant cell lines and investigate nilotinib, a second generation tyrosine kinase inhibitor (TKI), in preclinical models of GIST and imatinib-resistant GIST. For a model of imatinib-resistant GIST, we generated resistant cells from GK1C and GK3C cell lines by exposing them to imatinib for 6 months. The parent cell lines GK1C and GK3C showed imatinib sensitivity with IC₅₀ of 4.59±0.97 µM and 11.15±1.48 µM, respectively. The imatinib-resistant cell lines GK1C-IR and GK3C-IR showed imatinib resistance with IC₅₀ values of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively. The phosphorylation status of key cell signaling pathways, receptor tyrosine kinase KIT (CD117), platelet-derived growth factor receptor alpha (PDGFRA) and downstream signaling kinases: serine-threonine kinase Akt (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) or the non-receptor tyrosine kinase: proto-oncogene tyrosine-protein kinase Src (SRC), was analyzed in established cell lines and ERK1/2 phosphorylation was found to be increased compared to the parental cells. Nilotinib demonstrated significant antitumor efficacy against GIST xenograft lines and imatinib-resistant GIST cell lines. Thus, nilotinib may have clinical potential for patients with GIST or imatinib-resistant GIST.     

Long noncoding RNA H19 promotes chemotherapy resistance in choriocarcinoma cells

Choriocarcinoma (CC) is a trophoblast tumor prone to early distant organ metastases. At present, the main treatment for CC is chemotherapy, but chemotherapy resistance readily occurs and leads to treatment failure. H19 is a long noncoding RNA, and its abnormal expression has been found in various tumors, including CC. H19 is also considered to be related to the drug resistance mechanism of the same cancers. To investigate the role of H19 in drug-resistant CC cells, the following experiments were designed. We used human CC cell line JEG-3 to establish cell lines resistant to methotrexate and 5-fluorouracil (JEG-3/MTX and JEG-3/5-FU) and detected the expression of H19 in JEG-3, JEG-3/MTX, JEG-3/5-FU cells, JEG-3 with MTX, and JEG-3 with 5-FU. We found that the expression of H19 in the JEG-3/MTX and JEG-3/5-FU cells were significantly higher than that in JEG-3 cells. JEG-3 cells were treated with MTX or 5-FU for and quantitative real-time polymerase chain reaction assay revealed that H19 messenger RNA expression increased. Furthermore, after H19 was knocked out, the drug resistance index of the JEG-3/MTX and JEG-3/5-FU cells decreased; the proliferation, migration, and invasion ability diminished significantly; and apoptosis increased significantly. Finally, we detected the total and phosphorylation protein expression of phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) in the JEG-3/MTX and JEG-3/5-FU cells. The total protein of PI3K, AKT, and mTOR in the H19 knockout resistant cells showed no significant change relative to those in the H19 non-knockout resistant cells, whereas the phosphorylated proteins of PI3K, AKT, and mTOR were significantly decreased. Phosphorylated proteins of PI3K, AKT, and mTOR in the JEG-3/MTX and JEG-3/5-FU cells were significantly higher than that in JEG-3 cells. After using inhibition of phosphorylated PI3K/AKT/mTOR, the proliferation, migration, and invasion ability of the JEG-3/MTX and JEG-3/5-FU cells diminished significantly; and apoptosis increased significantly. On the basis of the above experiments, we concluded that H19 is related to the drug resistance of CC, and the knockout of H19 can reduce the drug resistance of resistant CC cells; and decrease the proliferative, migratory, and invasive ability; and increase the apoptosis. PI3K/AKT/mTOR pathway might be involved in H19-mediated effects. H19 is expected to be a therapeutic target for the treatment of drug-resistant chorionic carcinoma.     

INPP4B restrains cell proliferation and metastasis via regulation of the PI3K/AKT/SGK pathway

Cervical cancer continues to be among the most frequent gynaecologic cancers worldwide. The phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) pathway is constitutively activated in cervical cancer. Inositol polyphosphate 4‐phosphatase type II (INPP4B) is a phosphoinositide phosphatase and considered a negative regulatory factor of the PI3K/AKT pathway. INPP4B has diverse roles in various tumours, but its role in cervical cancer is largely unknown. In this study, we investigated the role of INPP4B in cervical cancer. Overexpression of INPP4B in HeLa, SiHa and C33a cells inhibited cell proliferation, metastasis and invasiveness in CCK‐8, colony formation, anchorage‐independent growth in soft agar and Transwell assay. INPP4B reduced the expression of some essential proteins in the PI3K/AKT/SGK3 pathway including p‐AKT, p‐SGK3, p‐mTOR, phospho‐p70S6K and PDK1. In addition, overexpression of INPP4B decreased xenograft tumour growth in nude mice. Loss of INPP4B protein expression was found in more than 60% of human cervical carcinoma samples. In conclusion, INPP4B impedes the proliferation and invasiveness of cervical cancer cells by inhibiting the activation of two downstream molecules of the PI3K pathway, AKT and SGK3. INPP4B acts as a tumour suppressor in cervical cancer cells.     

Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

Objective

Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations.

Materials and Methods

Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples.

Results

Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway.

Conclusion

Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways.     

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.