GATA3 Protein Expression Research Articles

Implication of GATA4 synonymous variants in congenital heart disease: A comprehensive in-silico approach

Synonymous variations, previously considered as neutral, are recently shown to have a significant impact on mRNA structure and stability thereby affecting protein expression and function. Their role in disease pathogenesis is also emerging. GATA4 is an important transcription factor involved in cardiac development and a well-known candidate gene associated with congenital heart disease (CHD). In the present study, we sought to conduct molecular screening of GATA4 gene in 285 sporadic and non-syndromic CHD cases. We identified four synonymous (c.27C>A, c.822C>T, c.1233G>A and c.1263C>T) and two intronic variants (g.83217T>G & g.85012T>A) in GATA4. Extensive computational analysis using widely acceptable tools i.e., Mfold, Human Splicing Finder (HSF) and Codon Usage bias was performed with a view to understand their putative downstream effects on GATA4 function. Mfold, a mRNA structure prediction tool showed the alterations of the mRNA structure and stability due to synonymous variants. Similarly, HSF also confidently predicted effect on the cis-acting regulatory elements of splicing due to four synonymous and one donor site intronic variants. Additionally, a significant change in ‘Relative Synonymous Codon usage (RSCU) frequencies’ and ‘log ratio of codon usage frequencies’ of variant codon was also noted that might affect the rate of translation. This study establishes that the synonymous variants are possibly associated with disease phenotype in CHD patients. Comprehensive computational analysis, using well-established web based tools, is suggestive of their potential downstream molecular effects on the structure, stability and expression of GATA4 protein.

Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome. The disease usually presents within the first year of life and is associated with anemia, congenital abnormalities, and an increased risk of developing cancer. The most prevalent mutations are found in the ribosomal protein RPS19, accounting for over 25% of cases but approximately 70% of DBA patients carry mutations in ribosomal genes. Downregulation of GATA1 has been attributed to the disease, but the upstream mechanisms leading to aberrant erythropoiesis is only beginning to be elucidated.SATB1 is highly upregulated in lymphocytes (especially thymocytes) and steadily downregulated in all myeloid ineages during differentiation. However the more modest expression of SATB1 in early progenitors is required for self-renewal of HSCs and globin switching in early erythropoiesis. Using transcriptomics, we identified SATB1 expression is prematurely lost in RPS19-insufficient erythropoiesis and that over a third (16/42) of RPS19-sensitive early erythroid genes were rescued upon SATB1 re-expression. One of the most deregulated transcripts encode the chaperone HSP70.GATA1 is an essential master regulator in erythroid differentiation and high GATA1 protein levels are required for efficient erythropoiesis. In DBA, GATA1 protein expression is drastically diminished. Two mechanisms contribute to this. At the translational level, GATA1 transcripts fail to be translated adequately due to ribosomal inefficiency at binding short, unstructured 5'UTRs. At the protein level,HSP70 loss leads to reduced GATA1 protein stability. During healthy erythropoiesis HSP70 genes are drastically upregulated (3.21, 4.18 and 1.37- fold) and this is lost in RPS19 insufficiency. Here we report that upregulation of HSP70 in healthy erythropoiesis requires SATB1. SATB1 binds to 3 sites within a 40kb region surrounding the HSP70 gene loci and recruits a distal predicted enhancer element to the proximal promoters via formation of two chromatin loops. During RPS19-insufficiency this chromatin organization is lost, but SATB1 re-expression restores chromatin looping and HSP70 expression, stabilizing GATA1 and promoting efficient erythropoiesis. As with HSP70, it is likely that permissive chromatin organization is essential for other RPS19-sensitive erythroid genes rescued by SATB1 re-expression. DisclosuresNo relevant conflicts of interest to declare.

The impact of the effectiveness of GATA3 as a prognostic factor in breast cancer

The transcription factor GATA3 plays a significant role in mammary gland development and differentiation. We analyzed expression of GATA3 in breast cancer (BC) cell lines and clinical specimens from BC patients in Taiwan. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR were carried out to determine the mRNA level of GATA3 from 241 pairs of matched tumor and adjacent normal tissues from anonymous female donors. GATA3 immunohistochemistry (IHC) staining and H-score were performed (n = 25). Inducing and silencing of GATA3 were done by exposure MCF-7 cell line to nicotine or curcumin, respectively. GATA3 expression was detected in most of the estrogen receptor-positive (ER+) tumor specimens (176/241, 73%) compared with paired normal tissues (65/241, 27%) (P < .001). The GATA3 level was highest in Luminal A, and independent t-tests revealed higher GATA3 was associated with ER+ (P = .018) and BC stages (stage II, and stage IV). Nuclear protein expression of GATA3 was detected in tumor tissues (P < .001) with higher H-score in Luminal A patients (P = .012). Kaplan-Meier survival analyses showed that ER+/progesterone receptor (PgR)+ and lower grade BC patients with relatively high GATA3 had better clinical overall survival (OS). GATA3 regulates ERα and BCL-2 as BC luminal subtype markers. Cox univariate and multivariate analyses demonstrated that the expression of GATA3 was an effective predictor of the risk of death. We demonstrated a correlation between GATA3 expression and only ER+ and suggest that a higher GATA3 expression is a good prognostic factor for OS for ER+ BC patients.

Application of GATA-3 gene marker in the detection of hematologic disorders in children.

The aim of the present study was to investigate the use of GATA-3 markers in the detection of hematologic disorders in children. In total, 35 pediatric patients diagnosed with blood disease and treated in Henan Red Cross Blood Center from January 2014 to June 2015 were selected for the observation group. Another 32 healthy children were selected for the control group. The differences in the GATA-3 mRNA expression levels between the control and observation groups were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The differences in the GATA-3 protein expression levels were detected via enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Compared with those in the healthy children, the mRNA expression levels of GATA-3 in patients with hematologic malignancies, acute lymphoblastic leukemia, myeloproliferative disorder, acute non-lymphocytic leukemia or thrombocytopenic purpura were significantly higher, and there were statistically significant differences between the groups (P<0.05). The results of ELISA showed that the GATA-3 protein expression levels in patients with hematologic malignancies (241.3±42.6 µg/l), acute lymphoblastic leukemia (196.3±21.6 µg/l), myeloproliferative disorder (284.2±45.1 µg/l), acute non-lymphocytic leukemia (269.3±31.4 µg/l) or thrombocytopenic purpura (272.1±39.1 µg/l) were significantly higher than those in healthy subjects (69.3±15.2 µg/l). The results of western blot analysis were consistent with those of ELISA. Based on our results, the expression levels of GATA-3 in healthy children and pediatric patients with blood diseases exhibit significant differences, and can be used as important markers for the clinical diagnosis of blood diseases in children.

Down-regulated GATA-1 up-regulates interferon regulatory factor 3 in lung adenocarcinoma

Interferon regulatory factor 3 (IRF-3) is widely known for its prompt response against viral infection by activating the interferon system. We previously reported that E2F1, Sp1 and Sp3 regulated transcriptional activity of IRF-3. Recently, different expression patterns of IRF-3 were found in lung cancer, leading to the alternation of the immunomodulatory function in tumorigenesis. However, the mechanism of transcriptional regulation of IRF-3 in lung cancer has not been extensively studied. Here, we investigated the characterization of IRF-3 promoter and found that GATA-1 bound to a specific domain of IRF-3 promoter in vitro and in vivo. We found elevated IRF-3 and decreased GATA-1 gene expression in lung adenocarcinoma in Oncomine database. Additionally, higher IRF-3 gene expression was observed in human lung adenocarcinoma, accompanied by aberrant GATA-1 protein expression. We further analyzed the relationship of GATA-1 and IRF-3 expression in lung adenocarcinoma cell lines and found that inhibition of GATA-1 by siRNA increased the promoter activity, mRNA and protein levels of IRF-3, while over-expression of GATA-1 down-regulated IRF-3 gene expression. Taken together, we conclude that reduced GATA-1 could be responsible for the upregulation of IRF-3 in lung adenocarcinoma cells through binding with a specific domain of IRF-3 promoter.

Effects of the multikinase inhibitor regorafenib in neuroblastoma.

10553 Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, and children with high-risk NB have poor survival rates and need novel treatment strategies. Regorafenib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for treating adult solid tumors such as advanced metastatic colorectal cancer and gastrointestinal stromal tumors, inhibits many RTKs, including PDGFR-β, VEGFR1-3, RET, c-Kit and FGFR family members. Based on the potential roles for these targets in neuroblastoma pathogenesis, we explored the therapeutic potential of Regorafenib alone and in combination with 13-cis-retinoic acid against neuroblastoma cells. Methods: We treated NB cell lines with increasing concentrations of Regorafenib and measured cell viability using MTT assays. We further measured the occupied percent confluence over time using continuous live cell imaging. We performed Western blots for caspase cleavage to measure apoptosis and flow cytometry to determine cell cycle expression. We performed Reverse Phase Protein Array (RPPA) analysis of neuroblastoma cells before and after treatment with regorafenib combined with 13- cis-retinoic acid. Results: IC50values for the tested cell lines ranged between 2.5mcM and 12.5mcM after 72 hours of exposure to Regorafenib, and decreased viability was due to a combination of apoptosis and cell cycle arrest. RPPA analysis identified alterations in multiple proteins and pathways after Regorafenib with retinoic acid treatment, including the PI3K/Akt/mTOR and Jak/Stat pathways. Phosphorylation of Erk1/2, S6, Akt, and c-Jun were decreased, while protein expression of GATA3 was increased in a dose-dependent manner. Conclusions: Regorafenib treatment results in reduced neuroblastoma cell viability and increased apoptosis via effects on several signaling pathways. Effects on intracellular signaling pathways associated with responses to the combination of regorafenib plus retinoic acid represent opportunities to develop novel combination therapies, representing potential new therapeutic strategies for children with neuroblastoma.

Mechanisms of Chinese Medicine Xinmailong\u2019s protection against heart failure in pressure-overloaded mice and cultured cardiomyocytes

Patients with heart failure (HF) have high mortality and mobility. Xinmailong (XML) injection, a Chinese Medicine, is clinically effective in treating HF. However, the mechanism of XML’s effectiveness on HF was unclear, and thus, was the target of the present study. We created a mouse model of pressure-overload-induced HF with transverse aortic constriction (TAC) surgery and compared among 4 study groups: SHAM (n = 10), TAC (n = 12), MET (metoprolol, positive drug treatment, n = 7) and XML (XML treatment, n = 14). Dynamic changes in cardiac structure and function were evaluated with echocardiography in vivo. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, AKT, GSK3β and protein expression of GATA4 in nucleus were detected with Western blot experiment. The results showed that XML reduced diastolic thickness of left ventricular posterior wall, increased ejection fraction and fraction shortening, so as to inhibit HF at 2 weeks after TAC. Moreover, XML inhibited the phosphorylation of ERK1/2, AKT and GSK3β, subsequently inhibiting protein expression of GATA4 in nucleus (P < 0.001). Together, our data demonstrated that XML inhibited the TAC-induced HF via inactivating the ERK1/2, AKT/GSK3β, and GATA4 signaling pathway.

Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis

AbstractTerminal erythroid differentiation is tightly coordinated with cell-cycle exit, which is regulated by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19INK4d, a member of CDKI family, is abundantly expressed in erythroblasts and that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, and led to increased apoptosis and generation of abnormally nucleated late-stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather, it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. Because the GATA1 protein is protected by nuclear heat shock protein family (HSP) member HSP70, we examined the effects of p19INK4d knockdown on HSP70 and found that p19INK4d knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced extracellular signal-regulated kinase (ERK) activation. Further biochemical analysis revealed that p19INK4d directly binds to Raf kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1, which in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19INK4d via a novel PEBP1-p-ERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.

Electro-acupuncture at Acupoint ST36 Ameliorates Inflammation and Regulates Th1/Th2 Balance in Delayed-Type Hypersensitivity.

Increasing evidence indicates anti-allergic and anti-inflammatory effects of electro-acupuncture (EA) therapy. However, its underlying mechanism on delayed-type hypersensitivity (DTH), a classic allergic inflammatory disease, still remains unclear. In this study, we aimed to explore the immunomodulatory mechanism of EA intervention in a mouse model of ovalbumin (OVA)-induced DTH. Mice were randomly divided into four groups: Control, OVA-DTH, DTH + EA, DTH + Sham. "Zusanli" acupoint (ST36) was used for DTH + EA, whereas a non-acupoint (localized 5mm below the "Zusanli" acupoint) was selected for DTH + Sham. Footpad thickness was checked, and the infiltration of inflammatory cells was estimated by hematoxylin and eosin staining. Levels of IgG and IgE in serum of different groups and inflammatory cytokines in the supernatants from homogenized footpads, including IFN-γ, TNF-α, IL-4, and IL-5, were determined by ELISA. Cell proliferation of spleen lymphocytes was assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). The frequency of CD4+IFN-γ+ and CD4+IL-4+ T cells was analyzed with flow cytometry. In addition, the mRNA and protein expression of T-bet and GATA-3 were evaluated by real-time PCR and Western blotting, respectively. Our data showed EA treatment at acupoint ST36 relieved the pathological progression of DTH responses via reduction in footpad swelling, infiltration of inflammatory cells, levels of IgG and IgE as well as decreased production of IFN-γ and TNF-α in homogenized footpad tissue. Moreover, detailed studies were performed revealing that EA attenuated the percentage of CD4+IFN-γ+ T cells and prevented Th cells differentiation into Th1 cells, and this results from inhibiting secretion of IFN-γ and suppressing expression of T-bet, an IFN-γ transcription factor. The results indicated that EA treatment improved Th1-mediated allergic skin inflammation via restoring Th1/Th2 balance by curbing Th1 differentiation. These findings suggested that EA at acupoint ST36 might be a useful and promising therapeutic for allergic inflammatory as well as Th1-mediated inflammation response.

Salvianolic Acid B Alleviates Heart Failure by Inactivating ERK1/2/GATA4 Signaling Pathway after Pressure Overload in Mice

BackgroundHeart failure(HF) is a dangerous disease that affects millions of patients. Radix Salvia is widely used in Chinese clinics to treat heart diseases. Salvianolic acid B(SalB) is the major active component of Radix Salvia. This study investigated the mechanisms of action and effects of SalB on HF in an experimental mouse model of HF.MethodsWe created a mouse model of HF by inducing pressure overload with transverse aortic constriction(TAC) surgery for 2 weeks and compared among 4 study groups: SHAM group (n = 10), TAC group (n = 9), TAC+MET group (metprolol, positive drug treatment, n = 9) and TAC+SalB group (SalB, 240 mg•kg-1•day-1, n = 9). Echocardiography was used to evaluate the dynamic changes in cardiac structure and function in vivo. Plasma brain natriuretic peptide (BNP) concentration was detected by Elisa method. In addition, H9C2 rat cardiomyocytes were cultured and Western blot were implemented to evaluate the phosphorylation of ERK1/2, AKT, and protein expression of GATA4.ResultsSalB significantly inhibited the phosphorylation of Thr202/Tyr204 sites of ERK1/2, but not Ser473 site of AKT, subsequently inhibited protein expression of GATA4 and plasma BNP(P < 0.001), and then inhibited HF at 2 weeks after TAC surgery.ConclusionsOur data provide a mechanism of inactivating the ERK1/2/GATA4 signaling pathway for SalB inhibition of the TAC-induced HF.

MiR-126 affects the invasion and migration of glioma cells through GATA4

We aimed to explore the relationship between miR-126 and glioma. miR-126 was highly expressed in low-grade clinical glioma tissue samples but lowly expressed in high-grade ones (P < .01). A human endogenous miR-126 expression vector was constructed. The migration capacity of cells transfected with the vector significantly decreased (P < .05). Up-regulation of miR-126 suppressed GATA4 protein expression. After transfection, they slightly contracted and became ovally or spherically shaped. F-actin significantly reduced, and microfilaments shortened or disappeared. The number of membrane-penetrating cells significantly decreased (P < .01). miR-126 inhibits the migration and invasion of glioma cells, which may be linked to GATA4 as a target gene.

Characterization of patient-derived tumor xenografts (PDXs) as models for estrogen receptor positive (ER+HER2− and ER+HER2+) breast cancers

The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2− and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2− tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2− tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.

Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma.

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is a tight junction molecule associated with epithelial and endothelial barrier function. Overexpression of JAM-A is also closely associated with invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. However, little is known about the mechanism in overexpression of JAM-A in head and neck squamous cell carcinoma (HNSCC). In the present study, we found high expression of JAM-A at the protein and mRNA levels in HNSCC tissues, including those of the oropharynx, larynx, and hypopharynx, together with high protein expression of β-catenin, p63, ΔNp63 and GATA-3. Furthermore, in ELISA, a significant increase of soluble JAM-A in the sera of HNSCC patients was observed compared to healthy subjects. Knockdown of JAM-A by siRNA inhibited cell proliferation, invasion and migration in the HNSCC cell line Detroit562 in vitro. JAM-A expression in Detroit562 was increased via a distinct signal transduction pathway including NF-κB. Expression of JAM-A, β-catenin, p63 and ΔNp63 in Detroit562 was decreased under hypoxia. Knockdown of p63, ΔNp63 or GATA-3 by siRNAs reduced JAM-A expression in Detroit562. In primary cultured HNSCC cells in which CK7, p63, ΔNp63 and GATA-3 were detected, JAM-A expression was decreased by knockdown of p63 or ΔNp63. These results indicate that JAM-A is a biomarker of malignancy in HNSCC and that plasma soluble JAM-A may contribute to serum-based diagnosis of HNSCC. The mechanism of dysregulation of JAM-A via p63/GATA-3 is important in possible molecular targeted therapy for HNSCC.

Immune imbalance of regulatory T/type 2 helper cells in the pathogenesis of allergic rhinitis in children.

To determine the role of regulatory T/type 2 helper cell-mediated immune imbalance in the pathogenesis of allergic rhinitis and examine the association between clinical severity and regulatory T/type 2 helper cell-mediated immune imbalance. Levels of interleukins 4 and 5 and transforming growth factor β1, and expression of FOXP3 and GATA3 (which are functionally related to regulatory T and type 2 helper cells, respectively), were evaluated in 46 allergic rhinitis patients and 42 healthy subjects. Compared to controls, allergic rhinitis patients showed significantly higher interleukin 4 and 5 levels, but lower transforming growth factor β1 levels. Furthermore, FOXP3 messenger RNA expression was lower in allergic rhinitis patients, while GATA3 messenger RNA and protein expression was significantly higher. Regulatory T/type 2 helper cell ratio was inversely correlated with clinical symptom scores. Regulatory T/type 2 helper cell immune imbalance may contribute to allergic rhinitis development. These findings provide a new insight into disease pathogenesis and potential therapeutic approaches.

Modulation of T-Bet and GATA-3 expression in experimental autoimmune thyroiditis rats through ginsenoside treatment

Hashimoto’s thyroiditis (HT) is one of the most common organ-specific autoimmune diseases. Increasing evidence indicates that HT may be characterized by an imbalance in the helper T cell subsets Th1 and Th2. Traditional Chinese Medicine (TCM) considers HT as a chronic exhaustion disease, leading to deficiency of qi. In TCM, qi indicates the functional power of the organs of the human body; hence TCM recommends focusing the treatment of HT so as to increase qi production. Ginseng is a well-known herbal medicine exhibiting a variety of efficacies, its main function-being to generate qi. Ginseng’s principal active component is ginsenoside, and modern pharmacology has shown that ginsenoside demonstrates biphasic immunomodulatory effects that can be utilized for the treatment of immune disorders. Previous work demonstrated that ginsenoside has a therapeutic effect on HT, but its mechanism is unknown. Experimental autoimmune thyroiditis rats were produced in order to investigate whether ginsenoside can modulate Th1/Th2 imbalance, the direct objective being to examine modulation of IFN-γ and IL-4 by ELISA, and the gene and protein expression of T-bet and GATA-3 by real-time PCR and Western blot. IFN-γ levels were increased while IL-4 levels decreased in EAT rats; treatment with ginsenoside led to decreased peripheral blood IFN-γ levels, with low doses statistically significant. Ginsenoside produced a biphasic effect on IL-4, with low and moderate doses promoting and high doses inhibiting secretion. Both protein and mRNA levels of T-bet were markedly reduced, while GATA-3 was significantly increased by ginsenoside.

Effects of cold preservation on the expression of GATA in intrahepatic bile duct

Objective To investigate the effects of cold preservation on the expression of GATA in intrahepatic bile duct. Methods The intrahepatic bile duct tissues of SD rats were obtained by collagenase perfusion combined with mechanical separation. After being cut into fragments, the intrahepatic bile duct tissues were cultured in rat tail collagen gel for 48 hours before experiment. All the rats were divided into the control group, cold preservation 1 hour (CP1 h) group and cold preservation 12 hours (CP12 h) group. There were 5 rats in each group. The mRNA and protein expressions of GATA were detected by Real-Time polymerase chain reaction and Western blot. Measurement data with normal distribution were presented as ±s. Comparison among 3 groups was done by ANOVA and pairwise comparisons were done by LSD test. Results The mRNA expressions of GATA3, GATA4, GATA6 were detected, while the mRNA expressions of GATA1, GATA2 and GATA5 were undetectable in intrahepatic bile duct tissue of the control group.The mRNA expressions of GATA4 in the CP1 h group, CP12 h group and the control group were 0.72 ± 0.08, 0.56 ± 0.07 and 0.96 ± 0.06, with significant difference among the 3 groups ( F =38.981, P 0.05) .The protein expressions of GATA4 in the CP1 h group, CP12 h group and the control group were 0.78 ±0.07, 0.64 ±0.06 and 0.99 ±0.10, with significant difference among the 3 groups ( F =24.211, P <0.05) .The protein expression of GATA4 in the CP12 h group was significantly lower than that in the CP1 h group and the control group, and the protein expression of GATA4 in the CP1 h group was significantly lower than that in the control group ( P <0.05) .The protein expressions of GATA6 in the CP1 h group, CP12 h group and the control group were 0.90 ±0.04, 0.75 ±0.06 and 0.98 ±0.11, with significant difference among the 3 groups ( F =11.651, P <0.05) .The protein expression of GATA6 in the CP12 h group was significantly lower than that in the CP1 h group and the control group ( P <0.05). Conclusion The expressions of GATA4 and GATA6 in the intrahepatic bile duct tissues are decreased significantly after cold preservation, which indicate that GATA4 and GATA6 might be involved in the pathophysiological process of the bile duct after cold preservation. Key words: Ischemic-type biliary lesions; Cold preservation; GATA; Intrahepatic bile duct

Abstract S2-04: Comprehensive molecular characterization of invasive lobular breast tumors

Abstract Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer accounting for 10-15% of invasive breast tumors. ILC is typically ER+ and beyond the known mutation and/or loss of E-cadherin function, which contributes to a highly discohesive morphology, little is known about the additional mechanisms driving ILC tumorigenesis, or alterations that differentiate ILC from invasive ductal carcinomas (IDC). Methods A dataset of 817 breast tumors from the TCGA Project, including 490 IDC, 127 ILC and 88 samples with a mixed IDC-ILC histology, were profiled on six genomic platforms to develop a comprehensive atlas of mutational, epigenetic, transcriptional and proteomic data. Integrative genomic analyses, both supervised and unsupervised, of ILC tumors and across histological subtypes were performed to identify genomic drivers of ILC oncogenesis. Results Comprehensive multi-platform analyses identified distinct molecular events associated with ILC tumors. As expected, lack of E-cadherin protein, as determined by Reverse Phase Protein Array (RPPA), and CDH1 mRNA expression was uniformly observed in ILC cases associated with distinct alterations targeting CDH1. In addition to previously reported CDH1 and PIK3CA mutations, we identified a number of novel ILC-enriched recurrent mutations targeting PTEN, RUNX1, TBX3, and FOXA1. An increased incidence of PTEN inactivating events, both mutations and copy number changes, were identified in ILC (13%) compared to IDC ER+ (7%), which corresponded with altered PTEN protein expression. These alterations were largely mutually exclusive with PIK3CA mutations and correlate with increased Akt activation as evident by increased Akt phosphorylation (pS473 and pT308), thus identifying a potential therapeutic opportunity for ILC patients. GATA3 signaling, which regulates epithelial cell differentiation, is frequently altered in luminal/ER+ breast cancers. Our analyses determined GATA3 mutations are more frequent in IDC luminal tumors as compared to ILC (19 % vs 5%). ILC luminal tumors show significantly lower GATA3 protein expression, but a higher frequency of mutations in FOXA1 (9% vs 2% in Luminal IDC), a transcription factor required to promote ER transcriptional programs. Within ILC tumors, FOXA1 mutations were found to cluster into a specific region of the Forkhead (FK) DNA binding domain. A broader analysis of FOXA1 mutations in breast and prostate cancer confirm two specific hotspots in the FK domain and the C-terminal transactivation domain. Interestingly, these mutational classes are associated with distinct transcriptional changes suggesting different functional effects. Finally, mRNA-seq analyses identified three robust molecular subclasses that are characterized by distinct genetic, genomic and proteomic patterns, including an increased immune-related group (Class 2), as well as differences in prognosis. Conclusions In this study, we developed a comprehensive atlas of genomic alterations that reveals key molecular differences differentiating ILC (FOXA1) from IDC (GATA3) tumorigenesis, a potential therapeutic target for ILC (Akt), and novel ILC subclasses based on underlying biological events. These findings provide further insight into the molecular heterogeneity of ER+ breast cancer. Citation Format: Giovanni Ciriello, Michael L Gatza, Katherine A Hoadley, Hailei Zhang, Suhn K Rhie, Reanne Bowlby, Matthew D Wilkerson, Cyriac Kandoth, Michael McLellan, Andrew Cherniack, Peter W Laird, Chris Sander, Tari A King, Charles M Perou. Comprehensive molecular characterization of invasive lobular breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S2-04.

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth.

IntroductionThe transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.MethodsChromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.ResultsIn this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.ConclusionsIn the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0491-x) contains supplementary material, which is available to authorized users.

189 Disturbed Flow Promotes Endothelial Cell Injury Via the Induction of Developmental Genes

IntroductionAtherosclerosis, a disease of arteries that can cause heart attack and stroke, is influenced by local blood flow patterns which exert wall shear stress (WSS) on endothelial cells (EC). Low,...

Abstract 266: Disturbed Flow Promotes Endothelial Cell Injury via the Induction of Developmental Genes

Introduction: Atherosclerosis is influenced by blood flow patterns which exert wall shear stress (WSS) on endothelial cells (EC). Low oscillatory WSS promotes atherosclerosis by inducing EC apoptosis and activation, whereas high unidirectional WSS is protective. We used microarray technology coupled to computational fluid dynamics to study the transcriptome of EC at regions of the porcine aorta exposed to low, oscillatory or high, unidirectional WSS. The study revealed differential expression of multiple genes that co-ordinate embryonic development. We hypothesised that this gene set includes WSS-sensitive regulators of EC survival. Methods and Results: EC were isolated from distinct anatomical regions of the porcine aorta using collagenase (n=6), and quantitative RT-PCR revealed elevated expression of developmental genes (GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2) at the inner (low WSS) compared to the outer curvature (high WSS) (all p&lt;0.05). Similarly, en face staining demonstrated that expression of GATA4 protein in EC was higher at the inner compared to the outer curvature of the murine aortic arch (p&lt;0.05). Studies using cultured porcine aortic EC (n=7) or human umbilical vein EC (n=7) revealed that GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2 were induced in HUVEC or PAEC exposed to low, oscillatory WSS for 72h using either the orbital shaking (210 rpm; +/-WSS 4 dyn/cm2) or IBIDI™ (+/-5 dyn/cm2 at 2Hz) systems (all p&lt;0.05). The rate of caspase-3 activation was significantly higher in EC exposed to low, oscillatory WSS compared to high, unidirectional WSS (3.1% vs 0.7%; p&lt;0.0001; n=10). Silencing of GATA4, FZD5 and BMP2 significantly reduced apoptosis in EC exposed to low, oscillatory WSS (1.2%, 0.6% and 1.6%; p&lt;0.05). Conclusions: We conclude that WSS influences EC viability through the induction of GATA4, FZD5 and BMP2; molecules that have a well-defined role in embryonic development. Further work is required to define the molecular mechanisms that underlie the induction of apoptosis by these molecules. Our observations illuminate the molecular mechanisms that regulate the focal nature of vascular injury and atherosclerosis.