GATA Binding Protein Research Articles

LncRNA MIAT binding to GATA3 activates MAPK signaling pathway and influences bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) manifests in premature neonates with aberrant pulmonary function. Numerous long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of BPD. This study aims to elucidate the impact of the lncRNA myocardial infarction-associated transcript (MIAT) on the initiation and progression of BPD. Initially, BPD murine models were established through hyperoxia induction in newborn mice. Subsequently, MIAT and GATA binding protein 3 (GATA3) expression levels were assessed, and intravenous administration of short hairpin RNAs (shRNAs) targeting MIAT and GATA3 was performed. Pulmonary histological alterations were examined through histological staining. Levels of inflammatory mediators were quantified using enzyme-linked immunosorbent assay (ELISA) kits. The interaction between MIAT and GATA3 was scrutinized through RNA immunoprecipitation, RNA pull-down, and fluorescence in situ hybridization. The downstream mechanisms of GATA3 were explored using bioinformatics analysis. In summary, lncRNA MIAT exhibited elevated expression in the lung tissues of BPD-afflicted mice. MIAT localized to the nucleus and interacted with GATA3, thereby activating the mitogen-activated protein kinase (MAPK) pathway. Knockdown of MIAT or silencing of GATA3 attenuated the inflammatory response, deactivated the MAPK pathway, and ameliorated BPD symptoms in mice, on the other hand, p-Cresyl sulfate potassium can activate the MAPK signaling pathway and attenuates the effects of si-MIAT or si-GAT3. These improvements were characterized by enhanced alveolar differentiation and reduced glycogen and collagen deposition. In conclusion, lncRNA MIAT plays a pivotal role in activating the MAPK pathway and exacerbating hyperoxia-induced BPD in mice through the binding to GATA3. It's an important discovery for the pathogenesis of BPD and may provide some new treatment for infants diagnosed with BPD.

FOXO3 induces TUG1-mediated miR-375/GATA3 signaling axis to promote the survival of melanocytes in vitiligo.

Vitiligo is characterized by the depletion of melanocytes due to the activation of CD8+ T cells. Taurine-upregulated gene 1 (TUG1), a long noncoding RNA, is involved in melanogenesis. This study aimed to explore the role and mechanism of TUG1 in vitiligo. RT-qPCR and western blot analyses demonstrated decreased TUG1 levels and increased miR-375 levels in patients with vitiligo. MTT and transwell assays indicated that TUG1 upregulation facilitated melanocyte survival and inhibited CD8+ T cell migration. Dual luciferase reporter and chromatin immunoprecipitation assays verified that Forkhead box O3 (FOXO3) directly interacted with the TUG1 promoter, leading to the positive regulation of TUG1 expression. In addition, FOXO3 promoted melanocyte survival by enhancing the transcription of TUG1. Luciferase reporter assay and RNA immunoprecipitation assay confirmed that TUG1 upregulated GATA binding protein 3 (GATA3) expression by targeting miR-375. TUG1 facilitated melanocyte survival by regulating the miR-375/GATA3 axis. In vitiligo, melanocyte survival is promoted by the induction of the TUG1-mediated miR-375/GATA3 axis by FOXO3, which offers new therapeutic targets for vitiligo treatment.

New insights into the mechanisms underlying 5-hydroxymethylfurfural-induced suppression of testosterone biosynthesis in vivo and in vitro

5-hydroxymethylfurfural (HMF), produced by the Maillard reaction, can indicate thermal processes in food. Previous research has examined the cytotoxic, genotoxic, mutagenic, and carcinogenic characteristics of HMF and its derivatives in different organs. Nevertheless, there is currently no available evidence about the impact of HMF on male reproductive toxicity. In this study, the effects of HMF on testosterone biosynthesis in both mouse testis and TM3 Leydig cells were investigated. HMF was administered to mice at doses of 30 and 300 mg/kg/day for 21 days and to Leydig cells at concentrations of 0.1, 1, and 10 mM for 24 h. The mechanism of action of HMF on testosterone biosynthesis in both mouse testis and Leydig cells was revealed by measuring the amount of testosterone, 3′,5′-cyclic adenosine monophosphate (cAMP) levels, and the expression level of some important genes in the steroidogenic pathway. In addition, its effects on general testis were examined through histopathological evaluations. Upon examination of the results, it was observed that HMF had a significant impact on reducing testosterone and cAMP levels. Furthermore, HMF inhibited the expression of steroidogenic genes, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxy dehydrogenase, and 17β-hydroxy dehydrogenase, as well as transcription factors, such as steroidogenic factor-1, GATA binding protein-4, and nerve growth factor IB. HMF-administrated groups had germinal epithelium degradation, vacuolization, and disorders in the interstitial area. Consequently, it has been proven for the first time that HMF can damage the male reproductive system by detrimentally impacting the production of testosterone.

GSDMD Mediates Ang II-Induced Hypertensive Nephropathy by Regulating the GATA2/AQP4 Signaling Pathway.

Hypertensive nephropathy is a common complication of hypertension. However, no effective measures are currently available to prevent the progression of renal insufficiency. Gasdermin D (GSDMD) is a crucial mediator of pyroptosis that induces an excessive inflammatory response. In the present study, we aimed to determine the effect of GSDMD on the pathogenesis of hypertensive nephropathy, which may provide new insights into the treatment of hypertensive nephropathy. C57BL/6 (wild-type, WT) and Gsdmd knockout (Gsdmd-/-) mice were subcutaneously infused with angiotensin II (Ang II) via osmotic mini-pumps to establish a hypertensive renal injury model. Recombinant adeno-associated virus serotype 9 (AAV9) carrying GSDMD cDNA was used to overexpress GSDMD. Renal function biomarkers, histopathological changes, and inflammation and fibrosis indices were assessed. Transcriptome sequencing (RNA-seq) and cleavage under targets and mentation (CUT & Tag) experiments were performed to identify the downstream pathogenic mechanisms of GSDMD in hypertensive nephropathy. GSDMD was activated in the kidneys of mice induced by Ang II (P < 0.001). This activation was primarily observed in the renal tubular epithelial cells (P < 0.0001). GSDMD deficiency attenuated renal injury and fibrosis induced by Ang II (P < 0.0001), whereas Gsdmd overexpression promoted renal injury and fibrosis (P < 0.01). Mechanistically, GSDMD increased Ang II-induced GATA binding protein 2 (GATA2) transcription factor expression (P < 0.01). GATA2 also bound to the aquaporin 4 (Aqp4) promoter sequence and facilitated Aqp4 transcription (P < 0.001), leading to renal injury and fibrosis. Moreover, treatment with GI-Y1, an inhibitor of GSDMD, alleviated Ang II-induced renal injury and fibrosis (P < 0.01). GSDMD plays an important role in the development of hypertensive nephropathy. Targeting GSDMD may be a therapeutic strategy for the treatment of hypertensive nephropathy.

Exploring the molecular underpinnings of macrosomia in gestational diabetes mellitus: The role of EGFR signaling and placental syncytiotrophoblast

Macrosomia, which is frequently associated with gestational diabetes mellitus (GDM), is linked to maternal glycemic control during gestation. When GDM is complicated by macrosomia (GDMM), the placenta exhibits increased mass, underscoring its role as a critical nexus for maternal-fetal nutrient exchange. Despite this recognized correlation, the underlying mechanisms propelling placental hypertrophy have remained elusive. Our study leveraged single-cell RNA transcriptome sequencing of GDMM placental tissues to pinpoint the specific syncytiotrophoblast (SCT) subsets that regulate placental dimensions. Notably, we observed pronounced upregulation of the epidermal growth factor receptor (EGFR) and its corresponding ligands, with a particular emphasis on the autoregulatory cascade involving the glycoprotein hormone alpha subunit (CGA), EGFR, and the transcription factor GATA binding protein 2 (GATA2), as well as perturbations in hormonal homeostasis within the SCT. Furthermore, our cell interaction analysis revealed an enhanced interplay between myeloid cells and SCT3, augmenting the EGFR signaling pathway. These molecular exchanges underscore the pivotal role of the placental immune microenvironment in the etiology of macrosomia, shedding light on the pathophysiology of GDMM and paving the way for novel therapeutic approaches.

Evaluation of breast-specific marker expression in metastatic breast cancers: Correlation with subtype switch.

This study evaluates the utility of breast specific markers in identifying breast cancer subtypes within metastatic settings. The subtype alteration in metastatic disease and its consequent impact on breast-specific marker expression is also examined. GATA-binding protein 3 (GATA3), mammaglobin (MMG), transcriptional repressor GATA binding 1 (TRSP1) and SRY-box transcription factor 10 (SOX10) expression were assessed in a large cohort of metastatic breast cancer (MBC) cases and correlated with the characteristics of both MBC and primary breast cancer (PBC). GATA3 was the most sensitive in MBC (83.1%), followed by TRPS1 (77.0%), MMG (58.5%) and SOX10 (7.1%). This trend was consistent in hormonal receptor (HR)+ and HR- MBC. Combining GATA3/TRPS1 yielded the highest detection rates in the overall cohort (90.1%) and HR+ MBC (97.1%), while TRSP1/MMG was most effective in HR- (76.2%) and TN (71.1%) MBC. Marker expression did not correlate with metastatic site, except SOX10 in lung metastases (P = 0.031). Subtype discordance between MBC and PBC occurred in 43 cases (24.4%), with GATA3 expression in HR- MBC significantly linked to subtype discordance (P = 0.005). Conversely, SOX10 expression was significantly associated with subtype concordance in HR- and TNBC (P ≤ 0.003). Despite a higher expression of GATA3 in all HR- cases, TRSP1 outperformed GATA3 in detecting concordant HR- cases (64.0% versus 38.5%). TRPS1 and SOX10 were expressed in more than 50% of concordant TNBC cases. The expression of breast-specific markers is mainly determined by the PBC subtype. GATA3 retains high sensitivity in HR+ cancers, even after HR loss during metastasis. TRPS1 and SOX10 are identified as valuable markers in TNBC metastasis.

The role and mechanism of NRG1/ErbB4 in inducing the differentiation of induced pluripotent stem cells into cardiomyocytes

BackgroundWe aimed to investigate the effect and potential mechanism of enhancing Neuregulin1 (NRG1)/v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ErbB4) expression on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes.MethodsWe utilized CRISPR-CAS9 technology to knock in ErbB4 and obtained a single-cell clone IPSN-AAVS1-CMV-ErbB4 (iPSCs-ErbB4). Subsequently, we induced the differentiation of iPSCs into cardiomyocytes and quantified the number of beating embryoid bodies. Furthermore, quantitative real-time PCR assessed the expression of cardiomyocyte markers, including ANP (atrial natriuretic peptide), Nkx2.5 (NK2 transcription factor related locus 5), and GATA4 (GATA binding protein 4). On the 14th day of differentiation, we observed the α-MHC (α-myosin heavy chain)-positive area using immunofluorescent staining and conducted western blotting to detect the expression of cTnT (cardiac troponin) protein and PI3K/Akt signaling pathway-related proteins. Additionally, we intervened the iPSCs-ErbB4 + NRG1 group with the PI3K/Akt inhibitor LY294002 and observed alterations in the expression of cardiomyocyte differentiation-related genes.ResultsThe number of beating embryoid bodies increased after promoting the expression of NRG1/ErbB4 compared to the iPSCs control group. Cardiomyocyte markers ANP, Nkx2.5, and GATA4 significantly increased on day 14 of differentiation, and the positive area of α-MHC was three times that of the iPSCs control group. Moreover, there was a marked increase in cTnT protein expression. However, there was no significant difference in cardiomyocyte differentiation between the iPSCs-ErbB4 group and the iPSCs control group. Akt phosphorylation was significantly increased in the iPSCs-ErbB4 + NRG1 group. LY294002 significantly reversed the enhancing effect of NRG1/ErbB4 overexpression on Akt phosphorylation as well as the increase in α-MHC and cTnT expression.ConclusionsIn conclusion, promoting the expression of NRG1/ErbB4 induced the differentiation of iPSC into cardiomyocytes, possibly through modulation of the PI3K/Akt signaling pathway.

Myocardial infarction in rats was alleviated by MSCs derived from the maternal segment of the human umbilical cord.

Mesenchymal stem cells (MSCs) are safe and effective in treating myocardial infarction (MI) and have broad application prospects. However, the heterogeneity of MSCs may affect their therapeutic effect on the disease. We recently found that MSCs derived from different segments of the same umbilical cord (UC) showed significant difference in the expression of genes that are related to heart development and injury repair. We therefore hypothesized that those MSCs with high expression of above genes are more effective to treat MI and tested it in this study. MSCs were isolated from 3cm-long segments of the maternal, middle and fetal segments of the UC (maternal-MSCs, middle-MSCs and fetal-MSCs, respectively). RNA-seq was used to analyze and compare the transcriptomes. We verified the effects of MSCs on oxygen-glucose deprivation (OGD)-induced cardiomyocyte apoptosis in vitro. In vivo, a rat MI model was established by ligating the left anterior descending coronary artery, and MSCs were injected into the myocardium surrounding the MI site. The therapeutic effects of MSCs derived from different segments of the UC were evaluated by examining cardiac function, histopathology, cardiomyocyte apoptosis, and angiogenesis. Compared to fetal-MSCs and middle-MSCs, maternal-MSCs exhibited significantly higher expression of genes that are associated with heart development, such as GATA-binding protein 4 (GATA4), and myocardin (MYOCD). Coculture with maternal-MSCs reduced OGD-induced cardiomyocyte apoptosis. In rats with MI, maternal-MSCs significantly restored cardiac contractile function and reduced the infarct size. Mechanistic experiments revealed that maternal-MSCs exerted cardioprotective effects by decreasing cardiomyocyte apoptosis, and promoting angiogenesis. Our data demonstrated that maternal segment-derived MSCs were a superior cell source for regenerative repair after MI. Segmental localization of the entire UC when isolating hUCMSCs was necessary to improve the effectiveness of clinical applications.

Single-cell multiplex immunocytochemistry in cell block preparations of metastatic breast cancer confirms sensitivity of GATA-binding protein 3 over gross cystic disease fluid protein 15 and mammaglobin.

Metastatic breast cancers are frequently encountered in cytology and require immunocytochemistry (ICC). In this study, traditional and multiplex ICC (mICC) for GATA-binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP15), and mammaglobin (MMG) were performed with the aim of validating mICC in cell blocks, with further single-cell expression pattern analysis to identify the single markers and combinations of markers most sensitive in subtypes of breast cancer. GATA3, GCDFP15, and MMG were paired with OptiView 3,3'-diaminobenzidine and Ventana DISCOVERY Purple and Blue, respectively, with cyclical and serial staining. Bright-field imaging was performed with the Mantra 2 system and analyzed with the inForm Tissue Finder (Akoya Biosciences). Cell detection and phenotyping were further confirmed by two pathologists. In the 36 cases studied, traditional ICC and mICC demonstrated good concordance (kappa coefficient, >0.5; p<.01) at three cutoffs (1%, 5%, and 50%), except for GATA3 at the 1% cutoff. Single-marker positivity outnumbered double-marker positivity and the exceedingly rare triple-marker positivity (<3%). GATA3 was the leading single marker-positive phenotype in all breast cancer subtypes, except for MMG in estrogen receptor-positive, progesterone receptor-positive, and human epidermal growth factor receptor 2-positive (ER+/PR+/HER2+) breast cancers. Limited to two markers, GATA3/MMG included the greatest number of tumor cells for luminal breast cancers (ER+/PR+/HER2+, 60.6%; ER+/PR+/HER2+, 31.4%), whereas HER2-overexpressed breast cancers (27.4%) and triple-negative breast cancers (26.4%) favored the combination of GATA3/GCDFP15. For a single marker, GATA3 displayed the highest sensitivity. The addition of MMG for hormone receptor-positive breast cancers and GCDFP15 for hormone receptor-negative breast cancers further increased sensitivity. The low proportion of multimarker-positive cells suggested that the coexpression observed with traditional ICC is attributable to intratumoral heterogeneity, not genuine coexpression.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

Blocking GATA6 Alleviates Pyroptosis and Inhibits Abdominal Wall Endometriosis Lesion Growth Through Inactivating the PI3K/AKT Pathway.

Endometriosis is a benign gynecological disorder characterized by the abnormal presence of endometrium-like cells, referred to as ectopic tissue, located outside the uterine cavity. Beyond the abnormal proliferation of endometrium-like tissues within and beyond the pelvic cavity, compelling scientific evidence underscores the crucial involvement of the NOD-like receptor NLRP3 inflammasome and pyroptosis in the pathogenesis of EMS. Our investigation has revealed a striking upregulation of the endogenous protein GATA-binding protein 6 (GATA6) in abdominal wall EMS. Notably, the knockdown of GATA6 significantly impaired the viability and migratory potential of primary ectopic endometrial stromal cells (EESCs) while also inhibiting crucial markers of pyroptosis, such as NLRP3, the gasdermin D N-terminal fragment (GSDMD-N), and reactive oxygen species (ROS) levels within these cells. Delving deeper into the underlying mechanisms, we discovered that suppressing GATA6 mitigated the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in EESCs. The administration of 740 Y-P, an agonist of the PI3K/AKT pathway, mitigated the inhibitive actions of GATA6 knockdown on EESCs' growth, migration, and pyroptosis, highlighting the intricate crosstalk between GATA6 and this intricate signaling cascade. In vivo experiments corroborated these findings, demonstrating that reduced GATA6 expression effectively restrained the growth of endometrial lesions and concurrently suppressed pyroptosis, accompanied by a dampening of PI3K/AKT signaling within these lesions. In summary, our study underscores the pivotal role of GATA6 in modulating the growth and pyroptosis of abdominal wall EMS through its regulation of the PI3K/AKT signaling pathway. Silencing GATA6 emerges as a promising approach to alleviate pyroptosis and potentially offers a novel therapeutic angle for managing abdominal wall EMS.

PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.

The inner cell mass (ICM) of early mouse embryos is specified into epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA-binding protein 6 (GATA6) in combination with fibroblast growth factor (FGF)/extracellular-signal-regulated kinase (ERK) signaling are central actors in ICM fate choice. However, what initiates the specification of ICM progenitors into Epi or PrE and whether other factors are involved in this process has not been fully understood yet. Here, we show that phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) is constitutively active during preimplantation development. Using pharmacological inhibition, we demonstrate that PI3K/AKT enables the formation of a functional ICM capable of giving rise to both the Epi and the PrE: it maintains the expression of the TF NANOG, which specifies the Epi, and confers responsiveness to FGF4, which is essential for PrE specification. Our work thus identifies PI3K/AKT signaling as an upstream regulator controlling the molecular events required for both Epi and PrE specification.

Silencing TRIM8 alleviates allergic asthma and suppressing Th2 differentiation through inhibiting NF-κB/NLRP3 signaling pathway

Background and aimAllergic asthma is a primary type of asthma and characterized by T helper 2 (Th2) cells -mediated inflammation. Tripartite motif containing 8 (TRIM8) protein is involved in immunoreaction and inflammatory response in many diseases. However, its role in allergic asthma remains unclear. Medical databank showed that TRIM8 was increased in lung of ovalbumin (OVA)-challenged mice. This study aimed to elucidate the effects of TRIM8 on allergic asthma and Th2 development. MethodsAsthma were induced by OVA challenge in mice, and the adenovirus vector loaded with TRIM8 knockdown sequence was delivered into asthma mice by nasal inhalation. The percentage of Th2 cells in lung was assessed by flow cytometric analysis, and the contents of Th2 cytokines (interleukin (IL)-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF) were assessed with ELISA. In vitro Th2 induction was performed in CD4+ cells from mouse spleen, the expression of Th2 molecules (IL-4, IL-5 and GATA binding protein 3 (GATA3)) were measured by real-time PCR. In addition, the nuclear factor-kappa B (NF-κB)/nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) signaling was determined. ResultsTRIM8 was highly expressed in the lung tissues of asthmatic mice and Th2-induced CD4+ cells. OVA challenge-induced Th2 development and Th2 cytokine secretion were restrained by silencing of TRIM8 in vivo. Similarly, the Th2 differentiation in vitro was also suppressed by TRIM8 knockdown. TRIM8 inhibited the NF-κB/NLRP3 activity by blocking transforming growth factor-beta-activated kinase 1 (TAK1), and the effects of TRIM8 were abrogated by overexpression of NLRP3. ConclusionsSilencing TRIM8 relieved the asthmatic injury in mice and excessive Th2 development via inhibiting the NF-κB/NLRP3 pathway. It is indicated that TRIM8 may contribute to the airway inflammation in allergic asthma via activating the NF-κB/NLRP3 signaling pathway. The current study provided a novel potential target for allergic asthma treatment.

GATA3 and markers of epithelial-mesenchymal transition predict long-term benefit from tamoxifen in ER-positive breast cancer

GATA binding protein 3 (GATA3) is essential for normal development of the mammary gland and associated with ER-positive breast cancer. Loss of GATA3 has been associated with epithelial-mesenchymal transition (EMT) in experimental studies. We investigated tumoral GATA3 in a cohort of postmenopausal patients with lymph-node negative breast cancer, randomized to adjuvant tamoxifen or control. Nuclear GATA3 expression was assessed with immunohistochemistry and GATA3 gene expression with Agilent microarrays. High GATA3 nuclear expression was associated with a lower rate of distant recurrence in ER-positive breast cancer (HR = 0.60, 95% CI 0.39–0.93). Low gene expression of GATA3 was associated with limited long-term benefit from adjuvant tamoxifen (interaction: p = 0.033). GATA3 gene expression was associated with the epithelial markers CDH1 (E-cadherin) and FOXA1, whereas negatively associated with several mesenchymal markers. Low expression of CDH1 was associated with marginal tamoxifen benefit (HR = 0.80 (0.43–1.49)), whereas patients with higher expression showed a significant benefit (HR = 0.33 (0.20–0.55), interaction: p = 0.029). In ER-positive breast cancer, diminished expression of GATA3 is associated with markers of EMT and poor long-term benefit from tamoxifen.

TRPS1, a sensitive marker for different histological and molecular types of breast cancer

ObjectivesWe explored Trichorhinophalangeal syndrome type 1 (TRPS1) expression in special types of breast carcinoma, and analyzed the correlation between TRPS1 and androgen receptor (AR) expression in triple-negative breast cancer (TNBC).MethodsTRPS1 expression was analyzed in 801 patients with special types of breast carcinoma. A total of 969 TNBC were used to analyze the correlation between the expression of TRPS1 and AR. TRPS1 expression was evaluated in 1975 cases of breast cancer with different molecular types.ResultsA total of 801 special types of breast cancers were stained with TRPS1.TRPS1 was positive in 100% (63/63) of mucinous carcinoma, 100% (7/7) adenoid cystic carcinomas (4 classic adenoid cystic carcinomas and 3 solid-basaloid adenoid cystic carcinomas), 100% (4/4) tubular carcinomas, 100% (2/2) secretory carcinomas, and 99.59% (243/244) invasive lobular carcinomas, 99.26% (267/269) invasive micropapillary carcinomas, 97.44% (38/39) ER-positive neuroendocrine tumors, 94.44% (34/36) metaplastic breast carcinomas (MBCs), 63.73% (65/102) apocrine carcinomas. TRPS1 was negative in all triple-negative neuroendocrine carcinomas (0/7).TRPS1 was positive in 92.86% (26/28) of metastatic special types of breast cancer. TRPS1 and AR expression were analyzed in 969 cases of TNBC. 90.40% were positive for TRPS1, and 42.41% were positive for AR. A significant inverse correlation between TRPS1 and AR expression was shown in TNBC (p < .001). TRPS1 showed a higher positive rate (93.13%) in TNBC compared to GATA binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP-15) and forkhead box transcription Factor C 1 (FOXC1).ConclusionsIn conclusion, our study demonstrated that TRPS1 is a highly sensitive marker for most special types of breast carcinoma. TRPS1 was positive in 63.73% of apocrine carcinomas. TRPS1 and AR expression was inversely correlated in TNBC.

Influence of mesenchymal and biophysical components on distal lung organoid differentiation

BackgroundChronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.MethodsHuman PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC).ResultsWe observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation.ConclusionOur data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

GATA4 downregulation enhances CCL20-mediated immunosuppression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a deadly cancer with a high global mortality rate, and the downregulation of GATA binding protein 4 (GATA4) has been implicated in HCC progression. In this study, we investigated the role of GATA4 in shaping the immune landscape of HCC. HCC tumor samples were classified into "low" or "normal/high" based on GATA4 RNA expression relative to adjacent non-tumor liver tissues. The immune landscapes of GATA4-low and GATA4-normal/high tumors were analyzed using cytometry by time-of-flight, bulk/spatial transcriptomic analyses and validated by multiplex immunofluorescence. GATA4-low tumors displayed enrichment in exhausted programmed cell death protein 1+ T cells, immunosuppressive regulatory T cells, myeloid-derived suppressor cells, and macrophages, highlighting the impact of GATA4 downregulation on immunosuppression. Spatial and bulk transcriptomic analyses revealed a negative correlation between GATA4 and C-C Motif Chemokine Ligand 20 (CCL20) expression in HCC. Overexpressing GATA4 confirmed CCL20 as a downstream target, contributing to an immunosuppressive tumor microenvironment, as evidenced by increased regulatory T cells and myeloid-derived suppressor cells in CCL20-high tumors. Lastly, the reduced expression of GATA4 and higher expression of CCL20 were associated with poorer overall survival in patients with HCC, implicating their roles in tumor progression. Our study reveals that GATA4 downregulation contributes to an immunosuppressive microenvironment, driven by CCL20-mediated enrichment of regulatory T cells and myeloid-derived suppressor cells in HCC. These findings underscore the critical role of GATA4 reduction in promoting immunosuppression and HCC progression.

YAP1 regulates thrombopoiesis by binding to MYH9 in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is a complicated bleeding disease characterized by a sharp platelet reduction. As a dominating element involved in ITP, megakaryocytes (MKs) are responsible for thrombopoiesis. However, the mechanism underlying the dysregulation of thrombopoiesis that occurs in ITP remains unidentified. In this study, we examined the role of Yes-associated protein 1 (YAP1) in thrombopoiesis during ITP. We observed reduced YAP1 expression with cytoskeletal actin misalignment in MKs from patients with ITP. Using an experimental ITP mouse model, we showed that reduced YAP1 expression induced aberrant MK distribution, reduced the percentage of late MKs among the total MKs, and caused submaximal platelet recovery. Mechanistically, YAP1 upregulation by binding of GATA-binding protein 1 to its promoter promoted MK maturation. Phosphorylated YAP1 promoted cytoskeletal activation by binding its WW2 domain to myosin heavy chain 9, thereby facilitating thrombopoiesis. Targeting YAP1 with its activator XMU-MP-1 was sufficient to rescue cytoskeletal defects and thrombopoiesis dysregulation in YAP1+/− mice with ITP and patients. Taken together, these results demonstrate the crucial role of YAP1 in thrombopoiesis, providing potential for the development of diagnostic markers and therapeutic options for ITP.

Deciphering Breast Origin in Malignant Effusions: The Diagnostic Utility of an MGP, GATA-3, and TRPS-1 Immunocytochemical Panel

Introduction: Defining the origin of metastatic cancer is crucial for establishing an optimal treatment strategy, especially when obtaining sufficient tissue from secondary malignancies is limited. While cytological examination is often used in this diagnostic setting, morphologic analysis alone often fails to differentiate metastases derived from the breast from other primaries. The hormone receptor, human epidermal growth factor receptor-2, gross cystic disease fluid protein 15, and mammaglobin immunohistochemistry are often used to diagnose metastatic breast cancer. However, their effectiveness decreases in estrogen receptor (ER)-negative breast cancers, including the triple-negative breast cancer (TNBC) subtype. Methods: We conducted a comprehensive evaluation of GATA-binding protein 3 (GATA-3), trichorhinophalangeal syndrome type 1 (TRPS-1), and Matrix Gla Protein (MGP) immunochemistry across 140 effusion cytology specimens with metastatic adenocarcinoma derived from various primaries, including the breast, colon, pancreaticobiliary, lung, tubo-ovarian, and stomach. Results: The expression rates of these immunomarkers were significantly higher in metastatic cancers originating from the breast than other primaries. In TNBC, TRPS-1 (80.00%) and MGP (65.00%) exhibited higher positivity rates compared to GATA-3 (40.00%). Additionally, our data suggest that an immunohistochemical panel comprising MGP, GATA-3, and TRPS-1 significantly enhances the detection of metastatic breast cancer in effusion cytology specimens, including TNBC in particular. When considering dual-marker positivity, the diagnostic accuracy was found to be 89.29% across all breast cancer subtypes and 92.93% for TNBC. Conclusions: MGP appears to be a robust marker for identifying metastatic breast cancer in malignant effusions, especially TNBC. MGP notably enhances diagnostic accuracy when incorporated together with GATA-3 and TRPS-1 in an immunohistochemical panel.

Association of GATA3 expression in triple-positive breast cancer with overall survival and immune cell infiltration

Breast cancer remains a leading cause of cancer-related mortality among women, with triple-positive breast cancer (TPBC) being a particularly aggressive subtype. GATA binding protein 3 (GATA3) plays a crucial role in the luminal differentiation of breast epithelium and T-cell differentiation. However, the relationship between GATA3 and immune infiltration in TPBC remains unclear. This study collected and analyzed TPBC data from The Cancer Genome Atlas (TCGA), METABRIC, and GSE123845 databases. Univariate and multivariate Cox regression analyses, along with Kaplan–Meier survival analyses, were employed to assess the prognostic value of GATA3 and other clinical features. Subsequently, Gene Set Enrichment Analysis (GSEA) was conducted to explore the potential biological functions and regulatory mechanisms of GATA3 in TPBC. Additionally, ssGSEA analysis revealed the connection between GATA3 and immune infiltration. And the effects of neoadjuvant chemotherapy and immunotherapy on GATA3 expression were also explored. Finally, clinical samples were used to detect the relationship between GATA3 expression and tumor infiltrating lymphocyte (TIL) levels. Our results demonstrated that GATA3 was significantly overexpressed in TPBC tissues compared to normal tissues (P < 0.05). A positive correlation between GATA3 mRNA and protein levels was observed (R = 0.55, P < 0.05). Notably, high GATA3 expression was associated with poor overall survival (HR = 1.24, 95% confidence interval (CI) 1.25–11.76, P < 0.05). GSEA indicated significant enrichment of immune-related gene sets in low GATA3 expression groups. Furthermore, pathologic complete response (pCR) patients exhibited significantly lower GATA3 expression compared to residual disease (RD) patients. Mutation analysis revealed higher PIK3CA and TP53 mutation rates in high GATA3 expression groups. Finally, clinical validation data showed that the degree of TILs was significantly higher in the low GATA3 expression group. In conclusion, this study suggests that high GATA3 expression may be associated with poor prognosis and may reduce immune infiltration in TPBC.