Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

PRDM14 extinction enables the initiation of trophoblast stem cell formation.

Trophoblast stem cells (TSCs) can be chemically converted from embryonic stem cells (ESCs) in vitro. Although several transcription factors (TFs) have been recognized as essential for TSC formation, it remains unclear how differentiation cues link elimination of stemness with the establishment of TSC identity. Here, we show that PRDM14, a critical pluripotent circuitry component, is reduced during the formation of TSCs. The reduction is further shown to be due to the activation of Wnt/ß-catenin signaling. The extinction of PRDM14 results in the erasure of H3K27me3 marks and chromatin opening in the gene loci of TSC TFs, including GATA3 and TFAP2C, which enables their expression and thus the initiation of the TSC formation process. Accordingly, PRDM14 reduction is proposed here as a critical event that couples elimination of stemness with the initiation of TSC formation. The present study provides novel insights into how induction signals initiate TSC formation.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis. / 基于Tollæ ·å—ä½“4/é«“æ ·åˆ†åŒ–å› å­88/æ ¸è½¬å½•å› å­κB信号通路探讨针刺"è¶è ­ç©´"æ”¹å–„å˜åº”æ€§é¼»ç‚Žå¤§é¼ å ç–«ç‚Žæ€§ååº”çš„æœºåˆ¶.

OBJECTIVES: To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. METHODS: Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. RESULTS: After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. CONCLUSIONS: Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Effect of ADSCs on Th17/Treg and T-bet/GATA-3 in model mice with primary immune thrombocytopenia.

We aimed to observe the effects of adipose-derived mesenchymal stem cells (ADSCs) on T helper 17 (Th17)/regulatory T cells (Treg) and T-box transcription factor (T-bet)/GATA-binding protein 3 (GATA-3) in model mice with primary immune thrombocytopenia (ITP). 32 BALB/C mice were selected. ADSCs were isolated from 2 mice and cultured. The other 30 mice were randomly divided into the normal control group, the ITP model control group, and the ITP experimental group. Platelet count (PLT), Th17/Treg cells, related serum cytokines [interleukin-6 (IL-6), IL-17A, IL-10, and transforming growth factor ß1 (TGF-ß1)], T-bet and GATA-3 mRNA levels in peripheral blood mononuclear cells (PBMCs) in the 3 groups were detected. PLT and Treg in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). Th17 and Th17/Treg in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-6 and IL-17A levels, and T-bet mRNA levels in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-10 and TGF-ß levels, and GATA-3 mRNA levels in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). ADSCs can effectively regulate Th17/Treg balance and improve T-bet/GATA-3 mRNA expression levels in ITP model mice.

Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation.

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

Diagnostic utility and sensitivities of matrix Gla protein (MGP), TRPS1 and GATA3 in breast cancer: focusing on metastatic breast cancer, invasive breast carcinoma with special features, and salivary gland-type tumours.

Matrix Gla protein (MGP) and trichorhinophalangeal syndrome type 1 (TRPS1) have recently emerged as novel breast-specific immunohistochemical (IHC) markers, particularly for triple-negative breast cancer (TNBC) and metaplastic carcinoma. The present study aimed to validate and compare the expression of MGP, TRPS1 and GATA binding protein 3 (GATA3) in metastatic breast carcinoma (MBC), invasive breast carcinoma (IBC) with special features, including special types of invasive breast carcinoma (IBC-STs) and invasive breast carcinoma of no special type with unique features, and mammary and non-mammary salivary gland-type tumours (SGTs). Among all enrolled cases, MGP, TRPS1 and GATA3 had comparable high positivity for ER/PR-positive (p=0.148) and HER2-positive (p=0.310) breast carcinoma (BC), while GATA3 positivity was significantly lower in TNBC (p<0.001). Similarly, the positive rates of MGP and TRPS1 in MBCs (99.4%), were higher than in GATA3 (90.9%, p<0.001). Among the IBC-STs, 98.4% of invasive lobular carcinomas (ILCs) were positive for all three markers. Among neuroendocrine tumours (NTs), all cases were positive for TRPS1 and GATA3, while MGP positivity was relatively low (81.8%, p=0.313). In the neuroendocrine carcinoma (NC) subgroup, all cases were positive for GATA3 and MGP, while one case was negative for TRPS1. All carcinomas with apocrine differentiation (APOs) were positive for GATA3 and MGP, while only 60% of the cases demonstrated moderate staining for TRPS1. Among mammary SGTs, MGP demonstrated the highest positivity (100%), followed by TRPS1 (96.0%) and GATA3 (72.0%). Positive staining for these markers was also frequently observed in non-mammary SGTs. Our findings further validate the high sensitivity of MGP and TRPS1 in MBCs, IBC-STs, and breast SGTs. However, none of these markers are capable of distinguishing between mammary and non-mammary SGTs.

Regulation of T helper cell differentiation by the interplay between histone modification and chromatin interaction.

The development and function of the immune system are controlled by temporospatial gene expression programs, which are regulated by cis-regulatory elements, chromatin structure, and trans-acting factors. In this study, we cataloged the dynamic histone modifications and chromatin interactions at regulatory regions during T helper (Th) cell differentiation. Our data revealed that the H3K4me1 landscape established by MLL4 in naive CD4+ T cells is critical for restructuring the regulatory interaction network and orchestrating gene expression during the early phase of Th differentiation. GATA3 plays a crucial role in further configuring H3K4me1 modification and the chromatin interaction network during Th2 differentiation. Furthermore, we demonstrated that HSS3-anchored chromatin loops function to restrict the activity of the Th2 locus control region (LCR), thus coordinating the expression of Th2 cytokines. Our results provide insights into the mechanisms of how the interplay between histone modifications, chromatin looping, and trans-acting factors contributes to the differentiation of Th cells.

Expression of BCL2, TP53, FOXA1, and GATA3 in pTa bladder cancer recurrence.

OBJECTIVES: In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence. METHODS: We analyzed samples of 79 patients with the pTa stage of BC using a real-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue. RESULTS: Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had a significantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant. CONCLUSION: Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had a significantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.

Unraveling the Impact of Blood Transfusion on Transcription Factors Regulating T Helper 1, 2, 17 and Regulatory T cells.

T helper 1 (TH1) and TH2 lymphocytes are the most important components of the immune system affected by blood transfusion. This study aimed`` to evaluate the effect of blood transfusion on gene expression of transcription factors related to the development of TH1, TH2, TH17 and regulatory T cells (Tregs). In this cross-sectional study, 20 patients diagnosed with abdominal aortic aneurysms requiring surgical repair were studied from January 2018 to August 2020. We utilized real-time PCR to evaluate the expression of transcription factor genes associated with TH1, TH2, TH17, and Treg, namely T-box-expressed-in-T-cells (T-bet), GATA-binding protein 3 (GATA-3), retinoid-related orphan receptor (RORγt), and fork head box protein 3 (Foxp3), respectively. The sampling occurred before anesthesia, 24- and 72 hours post-transfusion, and at the time of discharge. The results showed that the T-bet gene expression, compared to the time before transfusion, was significantly decreased 24 hours after blood transfusion and upon discharge while GATA3 genes exhibited a significant reduction both 24 and 72 hours after the transfusion, as compared to the pre-transfusion levels and the time of patient discharge. The Foxp3 gene demonstrated an increase at all study stages, with a notable surge, particularly 72 hours after red blood cell (RBC) transfusion. Conversely, the expression of RORγt gene, consistently decreased throughout all stages of the study. RBC transfusion in abdominal aortic aneurysm patients altered the balance of transcription gene expression of TH1, TH2, TH17, and Treg cells.

The transcription factor GATA3 positively regulates NRP1 to promote radiation-induced pulmonary fibrosis.

Radiation-Induced Pulmonary Fibrosis (RIPF) frequently arises as a delayed complication following radiation therapy for thoracic cancers, encompassing lung, breast, and esophageal malignancies. Characterized by a relentless and irreversible accumulation of extracellular matrix (ECM) proteins within the lung parenchyma, RIPF presents a significant clinical challenge. While the modulation of gene expression by transcription factors is a recognized aspect in various pathologies, their specific role in the context of RIPF has been less clear. This study elucidates that ionizing radiation prompts the translocation of the transcription factor GATA3 into the nucleus. This translocation facilitates GATA3's binding to the NRP1 promoter, thereby enhancing the transcription and subsequent translation of NRP1. Further investigations demonstrate that the TGF-ß pathway agonist, SRI-011381, can mitigate the effects of NRP1 knockdown on epithelial-mesenchymal transition (EMT) and ECM deposition, suggesting a pivotal role of the GATA3/NRP1/TGF-ß axis in the pathogenesis of RIPF. In conclusion, our findings not only underscore the critical involvement of GATA3 in RIPF but also highlight the GATA3/NRP1/TGF-ß signaling pathway as a promising target for therapeutic intervention in RIPF management.

Diagnosis of verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) using CK17, SOX2 and GATA3 immunohistochemistry.

AIMS: Verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) is an HPV-independent, p53 wild-type lesion with distinct morphology and documented risk of recurrence and cancer progression. vaVIN is rare, and prospective distinction from non-neoplastic hyperplastic lesions can be difficult. CK17, SOX2 and GATA3 immunohistochemistry has emerging value in the diagnosis of HPV-independent lesions, particularly differentiated VIN. We aimed to test the combined value of these markers in the diagnosis of vaVIN versus its non-neoplastic differentials in the vulva. METHODS AND RESULTS: CK17, SOX2 and GATA3 immunohistochemistry was evaluated on 16 vaVINs and 34 mimickers (verruciform xanthoma, lichen simplex chronicus, lichen sclerosus, psoriasis, pseudo-epitheliomatous hyperplasia). CK17 was scored as 3+ = full-thickness, 2+ = partial-thickness, 1+ = patchy, 0 = absent; SOX2 as 3+ = strong staining ≥ 10% cells, 2+ = moderate, 1 + =weak, 0 = staining in < 10% cells; and GATA3 as pattern 0 = loss in < 25% basal cells, 1 = loss in 25-75% basal cells, 2 = loss in > 75% basal cells. For analysis, results were recorded as positive (CK17 = 3+, SOX2 = 3+, GATA3 = patterns 1/2) or negative (CK17 = 2+/1+/0, SOX2 = 2+/1+/0, GATA3 = pattern 0). CK17, SOX2 and GATA3 positivity was documented in 81, 75 and 58% vaVINs, respectively, versus 32, 17 and 22% of non-neoplastic mimickers, respectively; ≥ 2 marker positivity conferred 83 sensitivity, 88 specificity and 86% accuracy in vaVIN diagnosis. Compared to vaVIN, SOX2 and GATA3 were differentially expressed in lichen sclerosus, lichen simplex chronicus and pseudo-epitheliomatous hyperplasia, whereas CK17 was differentially expressed in verruciform xanthoma and adjacent normal mucosa. CONCLUSIONS: CK17, SOX2 and GATA3 can be useful in the diagnosis of vaVIN and its distinction from hyperplastic non-neoplastic vulvar lesions. Although CK17 has higher sensitivity, SOX2 and GATA3 are more specific, and the combination of all markers shows optimal diagnostic accuracy.

A Metabolic-Epigenetic Mechanism Directs Cell Fate and Therapeutic Sensitivity in Breast Cancer.

Over the past decade, studies have increasingly shed light on a reciprocal relationship between cellular metabolism and cell fate, meaning that a cell's lineage both drives and is governed by its specific metabolic features. A recent study by Zhang and colleagues, published in Cell Metabolism, describes a novel metabolic-epigenetic regulatory axis that governs lineage identity in triple-negative breast cancer (TNBC). Among the key findings, the authors demonstrate that the metabolic enzyme pyruvate kinase M2 (PKM2) directly binds to the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the nucleus to silence expression of a set of genes that includes the mitochondrial carnitine transporter SLC16A9. Perturbation of this metabolic-epigenetic regulatory mechanism induces a metabolic shift away from glycolysis and toward fatty acid oxidation. The ensuing influx of carnitine facilitates the deposition of the activating epigenetic mark H3K27Ac onto the promoter of GATA3, driving a committed luminal lineage state. Importantly, this metabolic-epigenetic axis represents a potentially targetable vulnerability for the treatment of TNBC, a subtype that currently lacks effective therapeutic strategies. These findings lend further support for the paradigm shift underlying our understanding of cancer metabolism: that a cellular fuel source functions not only to provide energy but also to direct the epigenetic regulation of cell fate.

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer.

BACKGROUND: The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. RESULTS: The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. CONCLUSIONS: AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Deregulation of miR-1245b-5p and miR-92a-3p and their potential target gene, GATA3, in epithelial-mesenchymal transition pathway in breast cancer.

BACKGROUND: MicroRNAs (miRNAs) are small molecules that have prominent roles in tumor development and metastasis and can be used for diagnostic and therapeutic purposes. This study evaluated the expression of miR-92a-3p and miR-1245b-5p and their potential target gene, GATA3 in patients with breast cancer (BC). MATERIALS AND METHODS: In the search for BC-related microRNAs, miR-124b-5p and miR-92a-3p were selected using Medline through PubMed, miR2disease, miRcancer and miRTarBase. Moreover, target gene GATA3 and their possible interaction in the regulating epithelial-mesenchymal transition (EMT) and invasion was evaluated using in silico tools including miRTarBase, TargetScan, STRING-db, and Cytoscape. The expression level of miR-92a-3p, miR1245b-5p, and GATA3 were assessed on extracted RNAs of tumor and nontumor tissues from 36 patients with BC using qPCR. Additionally, clinical-pathologic characteristics, such as tumor grade, tumor stage, lymph node were taken into consideration and the diagnostic power of these miRNAs and GATA3 was evaluated using the ROC curve analysis. RESULTS: In silico evaluation of miR-92a-3p and miR-1245b-5p supports their potential association with EMT and invasion signaling pathways in BC pathogenesis. Comparing tumor tissues to nontumor tissues, we found a significant downregulation of miR-1245b-5p and miR-92a-3p and upregulation of GATA3. Patients with BC who had decreased miR-92a-3p expression also had higher rates of advanced stage/grade and ER expression, whereas decreased miR-1245b-5p expression was only linked to ER expression and was not associated with lymph node metastasis. The AUC of miR-1245b-5p, miR-92a-3p, and GATA3 using ROC curve was determined 0.6449 (p = .0239), 0.5980 (p = .1526), and 0.7415 (p < .0001), respectively, which showed a significant diagnostic accuracy of miR-1245b-5p and GATA3 between the BC patients and healthy individuals. CONCLUSION: MiR-1245b-5p, miR-92a-3p, and GATA3 gene contribute to BC pathogenesis and they may be having potential regulatory roles in signaling pathways involved in invasion and EMT pathways in BC pathogenesis, as a result of these findings. More research is needed to determine the regulatory mechanisms that they control.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

TRPS1 is a promising marker for all subtypes of breast cancer.

AIMS: Trichorhinophalangeal syndrome-1 (TRPS1) has been proposed as a novel breast marker with equally high expression in breast cancer (BC) subtypes, making it a useful diagnostic tool. Here, its expression was evaluated alongside other commonly used markers [GATA3, GCDFP15, mammaglobin (MGB) and SOX10] in a large cohort of BCs (n = 1852) and their corresponding nodal metastases. Its usefulness as a diagnostic tool and its correlation with clinicopathological features were assessed. METHODS AND RESULTS: TRPS1 was expressed at 75.8% overall in the BC cohort, with at least 58% expression among BC subtypes. It was less sensitive than GATA3 for luminal and HER2-overexpressing (HER2-OE) cancers (luminal A: 82 versus 97%; luminal B: 80 versus 95%; HER2-OE: 62 versus 76%), but it was the most sensitive for TNBC (60 versus ≤ 41%). It showed a stable expression in nodal metastases (primary tumour 76 versus nodal metastasis 78%), unlike a reduced nodal expression for GATA3 (86 versus 77%). TRPS1 outperformed GATA3 in detecting non-luminal cancers when paired with other breast markers. TRPS1 and GCDFP15 was the most sensitive combination in TNBC detection, with a 76% detection rate. For TRPS1-negative and GCDFP15-negative TNBCs, SOX10 was more sensitive than GATA3 (29 versus 24%). CONCLUSIONS: TRPS1 is a highly sensitive marker for all breast cancer subtypes, outperforming GATA3 in non-luminal cancers and displaying the highest sensitivity for TNBC detection when combined with GCDFP15. It is a valuable addition to the breast marker panel for accurate identification of BC.

TRPS1 and GATA3 Expression in Invasive Breast Carcinoma With Apocrine Differentiation.

CONTEXT.­: The recently identified immunohistochemical marker TRPS1 is highly sensitive and specific for invasive breast carcinoma, especially triple-negative breast carcinoma. However, TRPS1 expression in special morphologic subtypes of breast cancer is unclear. OBJECTIVE.­: To investigate the expression of TRPS1 in invasive breast cancer with apocrine differentiation, in comparison to the expression of GATA3. DESIGN.­: A total of 52 invasive breast carcinomas with apocrine differentiation, comprising 41 triple-negative breast carcinomas and 11 estrogen receptor (ER) and progesterone receptor (PR)-negative, human epidermal growth factor receptor 2 (HER2)-positive cases, along with 11 triple-negative breast carcinomas without apocrine differentiation, were evaluated for TRPS1 and GATA3 expression by immunohistochemistry. All tumors were diffusely positive (>90%) for androgen receptor (AR). RESULTS.­: Triple-negative breast carcinoma with apocrine differentiation had positive TRPS1 expression in 12% of cases (5 of 41), whereas GATA3 was positive in all cases. Similarly, HER2+/ER- invasive breast carcinoma with apocrine differentiation showed positive TRPS1 in 18% of cases (2 of 11), whereas GATA3 was positive in all cases. In contrast, triple-negative breast carcinoma with strong AR expression but without apocrine differentiation showed both TRPS1 and GATA3 expression in 100% (11 of 11) of cases. CONCLUSIONS.­: Most ER-/PR-/AR+ invasive breast carcinomas with apocrine differentiation are TRPS1 negative and GATA3 positive, regardless of HER2 status. Therefore, TRPS1 negativity does not exclude breast origin in tumors with apocrine differentiation. A panel of TRPS1 and GATA3 immunostains can be helpful when the tissue origin of such tumors is clinically relevant.

TRPS1 expression in primary and metastatic prostatic adenocarcinoma, muscle invasive bladder urothelial carcinoma, and breast carcinoma: Is TRPS1 truly specific and sensitive for a breast primary?

Trichorhinophalangeal syndrome type 1 (TRPS1) has been reported to be a sensitive and specific immunohistochemical (IHC) marker for breast carcinomas, especially when determining primary site of origin. However, there is limited data on TRPS1 expression in prostate and bladder cancers. A two-phase study was performed with 1) an exploratory cohort analyzing TRPS1 gene alterations in prostate, bladder, and breast carcinoma and TPRS1 mRNA expression data in prostate and bladder carcinoma; and 2) TRPS1 and GATA3 IHC in a confirmatory cohort in prostate, bladder, and breast carcinoma samples. Gene alterations were identified in a subset of breast, bladder, and prostate carcinomas and mRNA was consistently detected. In the IHC cohort, 183/210 (87.1 %) of breast, 22/69 (31.9 %) of prostate, and 20/73 (27.4 %) of urothelial carcinomas showed staining with TRPS1. Intermediate to high expression of TRPS1 was observed in 173/210 (82.8 %) of breast, 17/69 (24.6 %) of prostate, and 15/73 (20.5 %) of urothelial carcinomas. Furthermore, in prostate cancer, 26.9 % of pelvic lymph node metastases and 50 % in sites of distant metastases showed expression. Increased TRPS1 mRNA expression (p = 0.032) and IHC expression (p = 0.040) correlated with worse overall survival in bladder cancer. By comparison, GATA3 IHC stained 136/210 (64.8 %) of breast, 0/69 (0 %) of prostate, and 63/73 (93 %) of bladder carcinomas. Intermediate to high expression of GATA3 was seen in 131/210 (62.4 %) of breast and 63/73 (93 %) of bladder carcinomas. This study shows there is significant staining of TRPS1 in bladder and prostate cancers. As a result, comprehensive studies are needed to establish the true specificity of TRPS1 IHC stain across various tumor types before its widespread clinical adoption.

Aberrant expression of GATA3 in metastatic adenocarcinoma of the prostate: an important pitfall.

AIMS: The distinction of high-grade prostate cancer (PCa) from poorly differentiated urothelial carcinoma (UC) can be somewhat challenging on clinical and morphological grounds alone, yet it is of great importance for prognostication and choice of treatment. GATA3 is a useful immunohistochemical marker to confirm urothelial origin. However, recent works report strong GATA3 immunoexpression in primary high-grade PCa. The aim of this study was to explore GATA3 expression specifically in metastatic PCa. METHODS AND RESULTS: The pathology databases of four tertiary institutions were queried for cases of metastatic PCa. Available slides and clinical records were reviewed by experienced genitourinary pathologists. Prostatic markers (PSA, PSAP, NKX3.1) and GATA3 immunohistochemistry were performed. A total of 163 metastatic PCa cases were included. At least one prostate marker was positive in each case of non-regional distant metastasis, confirming prostatic origin. GATA3 strong staining was found in four (2.5%) cases: two liver, one bone and one non-regional lymph-node metastases. All four patients had Grade Group 5 PCa at the initial diagnosis. The metastatic prostatic adenocarcinomas were solid, either with no gland formation (n = 3) or with only focal cribriforming (n = 1). CONCLUSIONS: To our knowledge, this is the first study exploring GATA3 expression specifically in metastatic PCa. Despite being infrequent, GATA3 positivity in high-grade PCa may lead to misdiagnosis, with clinical implications. We recommend a panel of immunohistochemical markers, both prostatic and urothelial, for ruling out UC, either in primary tumour samples or in the event of metastases of unknown primary, when a genitourinary origin is suspected.

Analysis of GATA3 and FOXA2 expression suggests that downregulation of genes involved in the maintenance of a mature yolk sac tumor phenotype may underlie sarcomatoid transformation.

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3.