Forkhead Box A1 Research Articles

The ubiquitinase ZFP91 promotes tumor cell survival and confers chemoresistance through FOXA1 destabilization.

The forkhead box A1 (FOXA1), one of the forkhead class of DNA-binding proteins, functions as a transcription factor and plays a vital role in cellular control of embryonic development and cancer progression. Downregulation of FOXA1 has reported in several types of cancer, which contributes to cancer cell survival and chemoresistance. However, the mechanism for FOXA1 downregulation in cancer remains unclear. Here, we report that the ubiquitination enzyme zinc finger protein 91 (ZFP91) ubiquitinates and destabilizes FOXA1, which promotes cancer cell growth. High level of ZFP91 expression correlates with low level of FOXA1 protein in human gastric cancer (GC) cell lines and patient samples. Furthermore, ZFP91 knockdown reduces FOXA1 polyubiquitination, which decreases FOXA1 turnover and enhances cellular sensitivity to chemotherapy. Taken together, our findings reveal ZFP91-FOXA1 axis plays an important role in promoting GC progression and provides us a potential therapeutic intervention in the treatment of GC.

Forkhead‑boxA1 regulates tumor cell growth and predicts prognosis in colorectal cancer.

Forkhead boxA1 (FOXA1) functions as a tumor suppressor gene or an oncogene in various types of cancer; however, the distinct function of FOXA1 in colorectal cancer is unclear. The present study aimed to evaluate whether FOXA1 affects the oncogenic behavior of colorectal cancer cells, and to investigate its prognostic value in colorectal cancer. The impact of FOXA1 on tumor cell behavior was investigated using small interfering RNA and the pcDNA6‑myc vector in human colorectal cancer cell lines. To investigate the role of FOXA1 in the progression of human colorectal cancer, an immunohistochemical technique was used to localize FOXA1 protein in paraffin‑embedded tissue blocks obtained from 403patients with colorectal cancer. Tumor cell apoptosis and proliferation were evaluated using a terminal deoxynucleotidyl transferase‑mediated dUTP nick‑end labeling assay and Ki‑67 immunohistochemical staining, respectively. FOXA1 knockdown inhibited tumor cell invasion in colorectal cancer cells, and induced apoptosis and cell cycle arrest. FOXA1 knockdown activated cleaved caspase‑poly(ADP‑ribose) polymerase, upregulated the expression of p53 upregulated modulator of apoptosis, and downregulated BH3 interacting domain death agonist and myeloid cell leukemia‑1, leading to the induction of apoptosis. FOXA1 knockdown increased the phosphorylation level of signal transducer and activator of tran-scription‑3. By contrast, these results were reversed following the overexpression of FOXA1. The overexpression of FOXA1 was associated with differentiation, lymphovascular invasion, advanced tumor stage, depth of invasion, lymph node metastasis and poor survival rate. The mean Ki‑67 labeling index value of FOXA1‑positive tumors was significantly higher than that of FOXA1‑negative tumors. However, no significant association was observed between the expression of FOXA1 and the mean apoptotic index value. These results indicate that FOXA1 is associated with tumor progression via the modulation of tumor cell survival in human colorectal cancer.

Association of FOXA1 and EMT markers (Twist1 and E-cadherin) in breast cancer.

The transcriptionfactorFOXA1(forkhead box A1) plays key roles in tumor development and progression. In the present study, we analyzed the expression of FOXA1 in 52 breast tumors and 10 normal tissues, and investigated the relationship between FOXA1 and two EMT markers, namely Twist1 and E-cadherin by RT-PCR and IHC respectively. The expression level of FOXA1 was higher in tumor compared to normal tissues but the difference was not statistically significant (P = 0.138). FOXA1 expression correlated with less aggressive behavior as SBR grade I (P = 0.04), small tumors size (P = 0.05), and longer survival (P = 0.001). Furthermore, estrogen and progesterone positive tumors exhibit high level of FOXA1 (P = 0.002 and P = 0.038 respectively). Survival analysis showed that patients with ER positive/FOXA1 positive (P log rank = 0.001), PR positive/FOXA1 positive (P log rank = 0.044) and HER-2 negative/FOXA1 positive (P log rank = 0.002) tumors have a significant prolonged overall survival. On the other hand, the expression of E-cadherin positively correlated with FOXA1 (P = 0.028), whereas negative association was seen between the expression of Twist1 and FOXA1 (P = 0.016). Kaplan-Meier plots showed that patients with Twist1negative/FOXA1positive tumors have a significant prolonged overall survival (P log rank = 0.001) and FOXA1 appeared as independent predictors of patient survival in multivariate analyses. Overall, our results indicate that FOXA1 could be a useful biomarker to predict prognosis in breast cancer patients.

Functional Analysis of Promoter Variants in Genes Involved in Sex Steroid Action, DNA Repair and Cell Cycle Control.

Genetic variants affecting the regulation of gene expression are among the main causes of human diversity. The potential importance of regulatory polymorphisms is underscored by results from Genome Wide Association Studies, which have already implicated such polymorphisms in the susceptibility to complex diseases such as breast cancer. In this study, we re-sequenced the promoter regions of 24 genes involved in pathways related to breast cancer including sex steroid action, DNA repair, and cell cycle control in 60 unrelated Caucasian individuals. We constructed haplotypes and assessed the functional impact of promoter variants using gene reporter assays and electrophoretic mobility shift assays. We identified putative functional variants within the promoter regions of estrogen receptor 1 (ESR1), ESR2, forkhead box A1 (FOXA1), ubiquitin interaction motif containing 1 (UIMC1) and cell division cycle 7 (CDC7). The functional polymorphism on CDC7, rs13447455, influences CDC7 transcriptional activity in an allele-specific manner and alters DNA–protein complex formation in breast cancer cell lines. Moreover, results from the Breast Cancer Association Consortium show a marginal association between rs13447455 and breast cancer risk (p = 9.3 × 10−5), thus warranting further investigation. Furthermore, our study has helped provide methodological solutions to some technical difficulties that were encountered with gene reporter assays, particularly regarding inter-clone variability and statistical consistency.

Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody.

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

Upregulated forkhead-box A3 elevates the expression of forkhead-box A1 and forkhead-box A2 to promote metastasis in esophageal cancer.

Esophageal cancer (EC) is one of the most lethal cancers currently known. Members of the forkhead-box A (FOXA) family, including FOXA1 and FOXA2, have been reported to regulate EC progression. However, the role of FOXA3, which is another FOXA member, has not yet been investigated. In the present study, public dataset analyses and immunohistochemistry of 96 samples from patients with EC were performed to determine the potential roles of FOXA3 in EC. The results revealed that FOXA3 was significantly upregulated in EC tumor tissues and Barrett's esophagus tissues. In addition, FOXA3 upregulation was positively associated with tumor invasion, distant metastasis, tumor-node-metastasis stage and shorter overall survival in patients with EC, and multivariate analysis identified FOXA3 as an independent prognostic marker. In vitro experiments demonstrated that the migratory and invasive abilities of EC109 and EC9706 cell lines were inhibited following FOXA3 knockdown. Notably, FOXA3 expression levels were positively correlated with FOXA1 and FOXA2 expression levels according to The Cancer Genome Atlas dataset analysis. Furthermore, FOXA3 knockdown decreased the expression levels of FOXA1 and FOXA2 in EC109 and EC9706 cell lines. Conversely, FOXA1 or FOXA2 overexpression compensated for the effects of FOXA3 knockdown on the migratory and invasive capacities of EC cells. In conclusion, the present study demonstrated that FOXA3 upregulation in EC cells promoted metastasis through regulation of other FOXA members.

A novel lncRNA MCM3AP-AS1 promotes the growth of hepatocellular carcinoma by targeting miR-194-5p/FOXA1 axis

BackgroundHepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown.MethodsThe expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay.ResultsWe revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p’s target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells.ConclusionsOur results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

Epigenomic signatures in liver and blood of Wilson disease patients include hypermethylation of liver-specific enhancers

BackgroundWilson disease (WD) is an autosomal recessive disease caused by mutations in ATP7B encoding a copper transporter. Consequent copper accumulation results in a variable WD clinical phenotype involving hepatic, neurologic, and psychiatric symptoms, without clear genotype–phenotype correlations. The goal of this study was to analyze alterations in DNA methylation at the whole-genome level in liver and blood from patients with WD to investigate epigenomic alterations associated with WD diagnosis and phenotype. We used whole-genome bisulfite sequencing (WGBS) to examine distinct cohorts of WD subjects to determine whether DNA methylation could differentiate patients from healthy subjects and subjects with other liver diseases and distinguish between different WD phenotypes.ResultsWGBS analyses in liver identified 969 hypermethylated and 871 hypomethylated differentially methylated regions (DMRs) specifically identifying patients with WD, including 18 regions with genome-wide significance. WD-specific liver DMRs were associated with genes enriched for functions in folate and lipid metabolism and acute inflammatory response and could differentiate early from advanced fibrosis in WD patients. Functional annotation revealed that WD-hypermethylated liver DMRs were enriched in liver-specific enhancers, flanking active liver promoters, and binding sites of liver developmental transcription factors, including Hepatocyte Nuclear Factor 4 alpha (HNF4A), Retinoid X Receptor alpha (RXRA), Forkhead Box A1 (FOXA1), and FOXA2. DMRs associated with WD progression were also identified, including 15 with genome-wide significance. However, WD DMRs in liver were not related to large-scale changes in proportions of liver cell types. DMRs detected in blood differentiated WD patients from healthy and disease control subjects, and distinguished between patients with hepatic and neurologic WD manifestations. WD phenotype DMRs corresponded to genes enriched for functions in mental deterioration, abnormal B cell physiology, and as members of the polycomb repressive complex 1 (PRC1). 44 DMRs associated with WD phenotype tested in a small validation cohort had a predictive value of 0.9.ConclusionsWe identified a disease-mechanism relevant epigenomic signature of WD that reveals new insights into potential biomarkers and treatments for this complex monogenic disease.

Maintenance of the bladder cancer precursor urothelial hyperplasia requires FOXA1 and persistent expression of oncogenic HRAS

Tumorigenesis requires accumulation of genetic and epigenetic alterations, some of which drive tumor initiation. “Oncogene addiction” describes the phenomenon that (1) well-established cancers are dependent on one mutated oncogene or pathway for the maintenance of a malignant phenotype and that (2) withdrawal of the single oncogenic event leads to growth arrest and/or cancer regression. While oncogene addiction has been experimentally validated in advanced tumor models, its role in tumor precursors has not been investigated. We utilized the requirement of Forkhead box A1 (Foxa1) for transcriptional activation of the Upk2-promoter to temporally control the expression of Upk2-HRAS* oncogene, an inducer of urothelial hyperplasia in transgenic mice. Inducible homozygous knockout of Foxa1 in Upk2-HRAS*/UBC-CreERT2/Foxa1loxp/loxp mice results in reduced HRAS* levels. This led to a marked reduction of urothelial proliferation as evidenced by urothelial thinning, degenerative changes such as intracellular vacuole formation, and reduced Ki67 expression. Reduced proliferation did not affect basal, Krt14-positive cells, supporting the fact that Foxa1-regulated Upk2-HRAS* expression occurs primarily in supra-basal cells. Our results indicate that maintenance of urothelial hyperplasia in Upk2-HRAS* mice depends on continuous expression of Foxa1 and activated HRAS, and that mutated receptor tyrosine kinases, FOXA1 and/or other downstream effectors may mediate oncogene addiction in urothelial hyperplasia.

High Throughput Chemical Screening Reveals Multiple Regulatory Proteins on FOXA1 in Breast Cancer Cell Lines.

Forkhead box A1 (FOXA1) belongs to the forkhead class transcription factor family, playing pioneering function for hormone receptors in breast and prostate cancers, and mediating activation of linage specific enhancers. Interplay between FOXA1 and breast cancer specific signaling pathways has been reported previously, indicating a regulation network on FOXA1 in breast cancer cells. Here in this study, we aimed to identify which are the proteins that could potentially control FOXA1 function in breast cancer cell lines expressing different molecular markers. We first established a luciferase reporter system reflecting FOXA1 binding to DNA. Then, we applied high throughput chemical screening of multiple protein targets and mass spectrometry in breast cancer cell lines expressing different molecular markers: ER positive/HER2 negative (MCF-7), ER positive/HER2 positive (BT474), and ER negative/HER2 positive (MDA-MB-453). Regardless of estrogen receptor status, HER2 (human epidermal growth factor receptor 2) enriched cell lines showed similar response to kinase inhibitors, indicating the control of FOXA1 by cell signaling kinases. Among these kinases, we identified additional receptor tyrosine kinases and cyclin-dependent kinases as regulators of FOXA1. Furthermore, we performed proteomics experiments from FOXA1 inmunoprecipitated protein complex to identify that FOXA1 interacts with several proteins. Among all the targets, we identified cyclin-dependent kinase 1 (CDK1) as a positive factor to interact with FOXA1 in BT474 cell line. In silico analyses confirmed that cyclin-dependent kinases might be the kinases responsible for FOXA1 phosphorylation at the Forkhead domain and the transactivation domain. These results reveal that FOXA1 is potentially regulated by multiple kinases. The cell cycle control kinase CDK1 might control directly FOXA1 by phosphorylation and other kinases indirectly by means of regulating other proteins.

Expression and prognosis analyses of forkhead box A (FOXA) family in human lung cancer

Despite advances in early diagnosis and treatment, cancer still remains the major reason of mortality worldwide. The forkhead box A (FOXA) family is reported to participate in diverse human diseases. However, little is known about their expression and prognostic values in human lung cancer. Herein, we conducted a detailed cancer vs. normal analysis. The mRNA expression levels of FOXA family in numerous kind of cancers, including lung cancer, were analyzed using the Oncomine and GEPIA database. We observed that the mRNA expression levels of FOXA1, and FOXA3 were all increased while FOXA2 were decreased in most cancers compared with normal tissues, especially in lung cancer. Moreover, the expression levels of FOXA1, and FOXA3 are also highly expressed, while FOXA2 were decreased in almost all cancer cell lines, particularly in lung cancer cell lines, analyzing by Cancer Cell Line Encyclopedia (CCLE) and EMBL-EBI databases. Furthermore, the LinkedOmics database was used to evaluate the prognostic values, indicating that higher expression of FOXA1, FOXA3 indicated a poor overall survival (OS), while increased FOXA2 revealed a better OS in lung cancer. To conclusion, FOXA family showed significant expression differences between cancer and normal tissues, especially lung cancer, and FOXA1, FOXA3 could be promising prognostic biomarkers for lung cancer.

FOXA1 is upregulated in glioma and promotes proliferation as well as cell cycle through regulation of cyclin D1 expression.

IntroductionForkhead box A1 (FOXA1) has been found to upregulate in numerous cancers, such as ovarian cancer and glioma. However, the detailed function of FOXA1 in glioma is still not known. The purpose of this study was to explore the underlying mechanism of FOXA1 in glioma cell progression.MethodsThe expressions of FOXA1 in glioma tissues and cells were determined using quantitative reverse transcription-polymerase chain reaction and western blotting assays. Wound healing assay and Transwell invasion assay were employed to detect the effects of FOXA1 on cellular migration and invasion. Cell Counting Kit-8 assay, colony formation assay, and flow cytometry analyses were also performed.ResultsOur study results suggested FOXA1 was upregulated in glioma tissues and cells and revealed that FOXA1 promoted glioma cellular proliferation by facilitating G1/S transition. Previous work has indicated that CCND1 expression is regulated by FOXA1 in ovarian cancer. ChIP and qChIP assay as well as dual luciferase reporter assay validated that CCND1 expression was also regulated by FOXA1 in glioma cells. Moreover, over-expression of CCND1 in siFOXA1-transfected cells partly offsets the effect of FOXA1 inhibition on cellular proliferation.ConclusionFOXA1 promotes glioma cell progression, including cell proliferation and cell cycle, by targeting CCND1, and shows potential for the development of targeted treatment for glioma.

FOXA1 and AR in invasive breast cancer: new findings on their co-expression and impact on prognosis in ER-positive patients

BackgroundThe role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive (ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status.MethodsImmunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis.ResultsExpression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P < 0.001) and ER mRNA (r = 0.7326; P < 0.001). In ER+ BC, FOXA1 was associated with a good prognosis independently of AR expression in the three subgroups analyzed (FOXA1+/AR+; FOXA1+/AR-; FOXA1−/AR-). Multivariate analyses confirmed that FOXA1 may provide more information than AR in Disease-Free Interval (DFI) of ER+ BC patients.ConclusionOur results suggest that in BC the expression of FOXA1 is directly related to the expression of AR. Despite that, FOXA1 is found as superior predicting marker of recurrences compared to AR in ER+ BC patients.

Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine

Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for invitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared withmice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucosetolerance compared with administration of saline. In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of Lcells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.

Epigenetic Targeting of Aberrant Transcriptional Modulation in Pancreatic Cancer

While the mortality rates of cancer are generally declining, pancreatic cancer persists to be an exception with a 5-year-survival rate of less than 7%. Late diagnosis and resistance to conventional therapies contribute to high mortality rates in spite of the remarkable recent advances in cancer management and research. Consequently, there is an urgent need to find new and unconventional therapeutic targets to improve prognosis and survival of pancreatic cancer patients. In this review, we discuss the transcriptional effects of the most widely used epigenetic inhibitors in pancreatic cancer focusing on Bromodomain and Extraterminal domain (BET) and Histone Deacetylase (HDAC) inhibitors, which are currently highly promising therapeutic options. We suggest that these inhibitors can be better utilized at lower doses which exploit their transcriptional modulatory effects on pancreatic cancer transcriptional programs directed by specific factors such as MYC and Forkhead Box A1 (FOXA1), rather than simply based on their anti-proliferative effects. This approach can potentially help avoid the intolerable adverse events frequently elicited by the use of these treatments at higher doses. In particular, we underscore the crucial role of distal regulatory elements in mediating the specific effects of these epigenetic inhibitors and propose using them in a more selective and prudent manner.

Fluorescence sensing of protein-DNA interactions using conjugated polymers and graphene oxide

Protein-DNA binding, particularly transcription factor-DNA binding, is one of the main molecular interactions involved in gene regulation. These interactions are sequence-specific, play a key role in many fundamental biological processes, and are deregulated in the pathogenesis of several diseases. In this study, a robust analytical bioassay to characterize protein-DNA binding was built by combining the optical properties of water soluble conjugated polymers, and graphene oxide’s superquenching capabilities. Cationic conjugated polymers bind strongly to double stranded DNA through electrostatic interactions, and provide fluorescent signals to track the DNA without any chemical modification. In addition, the labeled DNA retains its protein binding ability. An important oncogenic transcription factor (i.e. estrogen receptor α) was used to demonstrate the concept, and two collaborative factors involved in the estrogen gene transcription (i.e. forkhead box A1 and activating enhancer binding protein 2 gamma) were employed as controls. This method overcame the main limitations of previous nanomaterial-based bioassays, while keeping the sensitivity and precision of the gold standard techniques. These benefits, combined with the high versatility and low-costs, could lead this bioassay to be used in several fundamental biomedical research lines, such as large scale protein-DNA binding studies and drug discovery.

Identification of Transmembrane Protease Serine 2 and Forkhead Box A1 As the Potential Bisphenol A Responsive Genes in the Neonatal Male Rat Brain.

Perinatal exposure of Bisphenol A (BPA) to rodents modifies their behavior in later life. To understand how BPA modifies their neurodevelopmental process, we first searched for BPA responsive genes from androgen and estrogen receptor signaling target genes by polymerase chain reaction array in the neonatal male rat brain. We used a transgenic strain of Wistar rats carrying enhanced green fluorescent protein tagged to gonadotropin-inhibitory hormone (GnIH) promoter to investigate the possible interaction of BPA responsive genes and GnIH neurons. We found upregulation of transmembrane protease serine 2 (Tmprss2), an androgen receptor signaling target gene, and downregulation of Forkhead box A1 (Foxa1), an ER signaling target gene, in the medial amygdala of male rats that were subcutaneously administered with BPA from day 1 to 3. Tmprss2-immunoreactive (ir) cells were distributed in the olfactory bulb, cerebral cortex, hippocampus, amygdala, and hypothalamus in 3 days old but not in 1-month-old male rats. Density of Tmprss2-ir cells in the medial amygdala was increased by daily administration of BPA from day 1 to 3. Tmprss2 immunoreactivity was observed in 26.5% of GnIH neurons clustered from the ventral region of the ventromedial hypothalamic nucleus to the dorsal region of the arcuate nucleus of 3-day-old male rat hypothalamus. However, Tmprss2 mRNA expression significantly decreased in the amygdala and hypothalamus of 1-month-old male rats. Foxa1 mRNA expression was higher in the hypothalamus than the amygdala in 3 days old male rats. Intense Foxa1-ir cells were only found in the peduncular part of lateral hypothalamus of 3-day-old male rats. Density of Foxa1-ir cells in the hypothalamus was decreased by daily administration of BPA from day 1 to 3. Foxa1 mRNA expression in the hypothalamus also significantly decreased at 1 month. These results suggest that BPA disturbs the neurodevelopmental process and behavior of rats later in their life by modifying Tmprss2 and Foxa1 expressions in the brain.

Novel transgenic knockout model of basal-squamous bladder cancer.

459 Background: The recent description of the highly aggressive Basal-Squamous molecular subtype of bladder cancer (BLCa) requires the development of new in vivo models for the study of this clinical entity. Although loss of the transcription factor Forkhead box A1 ( FOXA1) is significantly associated with the Basal-Squamous subtype, cooperating genetic alterations are unknown. Herein, we identify genetic alterations that potentially cooperate with FOXA1 loss to drive tumorigenesis and/or the Basal-Squamous phenotype and develop a novel transgenic model for the study of Basal-Squamous BLCa. Methods: We interrogated The Cancer Genome Atlas (TCGA) BLCa study to identify common genetic alterations associated with FOXA1 copy number (CN) loss. Based on our results, we utilized a bladder-specific Cre-LoxP ( Uroplakin II-Cre) to constitutively knock-out (KO) Foxa1 and/or Pten in mouse urothelium. Two cohorts of mice were aged for 6 and 12 months, and a third cohort was exposed to the bladder-specific carcinogen N-butyl-(4-hydroxybutyl)-nitrosamine (BBN; 0.05%) for 12 weeks. Following all experiments, the bladder tissue was isolated and characterized. Results: The TCGA data revealed that combined CN loss of both FOXA1 and PTEN occurs in 17% of MI BLCa patients. At 6 months of age, our mice developed urothelial hyperplasia, whereas at 12 months of age, double KO ( Foxa1-/-/Pten-/-) mice developed carcinoma in situ (CIS) with SqD, enriched for basal (Krt5/6 and 14) and reduced in luminal (Gata3 and Pparγ) markers. Increased tumor stage was significantly associated with combined KO of at least one allele of Foxa1 and Pten following BBN exposure. Moreover, upregulation of apoptosis related genes was observed by RNA-sequencing data. Conclusions: In advanced BLCa, CN loss of FOXA1 and PTEN is common. Genetic ablation of Foxa1 and Pten results in CIS with SqD and a pathologic profile consistent with the Basal-Squamous subtype and accelerated tumor onset following carcinogen exposure. Therefore, we describe the development of a novel model for the study of Basal-Squamous BLCa potentially useful for preclinical studies.

Clinical significance and prognostic value of Forkhead box A1 expression in human epithelial ovarian cancer.

Forkhead box (FOX) A1 is a member of the FOX family of transcription factors, which serve a function in numerous types of tumor. The present study assessed the potential role of FOXA1 in human epithelial ovarian carcinoma (EOC). Total RNA was isolated from 16 fresh-frozen EOC tumors with paired corresponding non-malignant ovarian epithelium tissues, and FOXA1 expression was analyzed using reverse transcription-quantitative polymerase chain reaction. Immunohistochemical analysis was performed to evaluate FOXA1 expression in 110 epithelial ovarian carcinoma tissue specimens (including 80 serous papillary adenocarcinoma, 9 clear cell carcinoma, 12 endometrioid adenocarcinoma, 5 mucinous carcinoma and 4 transitional cell carcinoma specimens), 24 benign ovarian tumor surface epithelium tissues and 10 normal ovarian tissue samples. The present study analyzed the association between FOXA1 expression and clinical characteristics in patients with EOC. The Kaplan-Meier method was used for survival analysis. The results of the present study revealed that FOXA1 mRNA expression was significantly increased in EOC tissues compared with paired normal ovarian samples (P=0.014). The immunohistochemical expression of FOXA1 in EOC tissues was associated with the FIGO grade, differentiation status and overall survival time (all P<0.05). Finally, the significance of FOXA1 expression in the prognosis of the patients was evaluated. The results of Kaplan-Meier survival curve revealed that high FOXA1 expression was associated with decreased overall survival time in the patients, relative to low FOXA1 expression (P=0.0132). In conclusion, FOXA1 is overexpressed in EOC and associated with clinicopathological features, including overall survival time. FOXA1 potentially represents a novel biomarker and therapeutic target for EOC.

Integrated Bioinformatics Analysis of Potential Biomarkers for Prostate Cancer.

The aim was to expound the pathogenesis of prostate cancer and to identify the potentially biomarkers for prostate cancer (PC). DNA methylation microarray data GSE38240 containing 8 prostate cancer metastases and 4 normal prostate samples as well as gene expression profile data GSE26910 containing 6 prostate primary tumors and 6 normal samples were used. Differentially expressed genes (DEGs) and differently methylated sites of PC were screened and the regulatory network was constructed with DEGs-related transcription factors (TFs). The obtained hub genes were subjected to protein-protein interaction network analysis. Enrichment analysis of down-regulated DEGs were performed. Total 351 DEGs including 190 down-regulated and 161 up-regulated genes and 3234 differently methylated sites were identified. In total 69 DEGs-related TFs were found. Regulatory network contained 1301 nodes and 2527 connection pairs and that FOXA1 (forkhead box A1), BZRAP1-AS1 (benzodiazapine receptor associated protein 1 antisense RNA 1) and KRT8 (keratin 8) were the top three nodes of it. The enriched GO terms were mainly biological activity of the blood and cells-related. Total 29 DEGs (such as AGTR1, angiotensin II receptor, type 1) and 57 none-DEGs involved in the PPI network. Biological functions in blood circulation and the involved AGTR1 may play important roles in PC by gene-methylation. Besides, BZRAP1-AS1 may be novel biomarker related with PC.