GATA3 Expression Research Articles

GATA3 Immunohistochemical Staining in Classical Hodgkin Lymphoma and its Diagnostic Utility in Differential Diagnosis.

Classical Hodgkin lymphoma (CHL) is a common lymphoid neoplasm with a wide range of differential diagnosis. Although it has a specific immunophenotype, aberrant expression of antigens can cause problems at its diagnosis. In this study we evaluated the usefulness of GATA3 in differential diagnosis of classical Hodgkin lymphoma. One hundred CHL cases and a control group of 106 lymphoma cases, which include anaplastic large cell lymphoma (ALK (+) and (-)), EBV (+) large B cell lymphoma, T-cell/ histiocyte rich B cell lymphoma, primary mediastinal large B cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma and mediastinal grey zone lymphoma, were included in the study. GATA3 immunohistochemistry were applied to all cases and its nuclear expression was accepted as positive. Expression status of GATA3 was compared in between the CHL and the control group, as well as among each lymphoma subtype. In addition, whether the biopsy type effects its diagnostic performance is assessed. In CHLs relationship with EBV status and GATA3 expression is evaluated. GATA3 expression was significantly higher in CHL cases compared to the control group (p<0,001). When compared with the individual subgroups GATA3 is still found to be useful in differential diagnosis except for ALK (-) ALCL (p=0,678) and mediastinal gray zone lymphomas (p=0,327). GATA3 expression is significantly higher in EBV (-) CHLs (p=0,02). In core needle biopsies its diagnostic performance is limited (p=0,178). GATA3 is a useful marker for differentiating CHL from B-cell non-Hodgkin lymphomas but its efficiency is limited in ALK (-) ALCL and mediastinal grey zone lymphomas. Due to the heterogeneous reaction diagnostic value is limited in core needle biopsies.

MiRNAs targeted transcription factors HaGATAa/b to mediate the post-mating switch in Helicoverpa armigera female reproductive behavior.

The process of mating induces significant shifts in female reproductive behavior across various species, with the postmating behavioral switch playing a crucial role in insect reproduction. Previous studies have demonstrated the regulatory role of GATA transcription factors in vitellogenin transcription and egg formation in insects, while miRNAs have been implicated in modulating GATA expression and insect reproductive processes. Nevertheless, the precise regulatory mechanism underlying the interaction between miRNAs and GATA transcription factors in the postmating behavioral switch remains largely unexplored. In this study, we identified two key GATA transcription factors, HaGATAa and HaGATAb, as central players in orchestrating the postmating behavior of H. armigera using transcriptomics and RNAi technologies. HaGATAa was found to act upstream of HaGATAb, regulating its expression. Furthermore, we observed a postmating increase in miR-282 levels in females, targeting HaGATAa to regulate egg-laying capacity. Conversely, the decreased expression of miR-2 following mating functioned as a negative feedback regulator, influencing the expression of HaGATAb and thus impacting the postmating behavior of female individuals. Our results revealed a signal-mediated feedback regulatory mechanism that sustains female postmating behavior in H. armigera. These findings not only establish a strong basis for understanding the postmating behavior mechanisms in female moths, but also offer valuable insights for identifying potential targets for pest control strategies. © 2024 Society of Chemical Industry.

Novel Expression of Apical Bile Acid Transport (ASBT) More Proximally Than Distal Ileum Contributing to Enhanced Intestinal Bile Acid Absorption in Obesity

Dietary lipid absorption is facilitated by bile acids. In the Zucker rat (ZR) model of obesity, bile acid absorption, mediated by the apical sodium bile acid transporter (ASBT), was increased in villus cells from the distal ileum. However, whether ASBT may be de novo expressed more proximally in the small intestine during obesity to facilitate additional bile acid absorption is not known. For this, starting from the end of the ileum to the mid jejunum, caudal-orally, five intestinal segments of equal length (S1–S5) were separated from lean and obese ZRs (LZR and OZR). Intestinal mucosa obtained from these segments were used for total RNA extraction, RT-qPCR and 3H-TCA uptake. The results showed that bile acid absorption along with the mRNA expression of ASBT and FXR progressively decreased caudal-orally in both LZRs and OZRs but was significantly higher in all small intestinal segments in OZRs. The expression of GATA4 was absent in the distal ileum (S1) in both LZRs and OZRs, but steadily increased along the proximal length in both. However, this steady increase was significantly reduced in the comparative obese proximal intestinal segments S2, S3, S4 and S5. The expressions of bile acid-activated G-protein-coupled bile acid receptor TGR5 and S1PR2 were unaltered in segments S1–S4 but were significantly increased in OZR S5. The paradigm changing observation of this study is that ASBT is expressed more proximally in the small intestine in obesity. This likely increases overall bile acid absorption and thereby lipid absorption in the proximal small intestine in obesity.

Immunohistochemical Analysis of GATA2 Expression in Endometrium and its Relationship with Hormone Receptor Expression in Benign and Premalignant Endometrial Disorders.

The GATA gene family encodes highly conserved zinc-finger transcription factors that facilitate the development and function of multiple organ systems including the uterus. In the endometrium, GATA2 functions in a positive autoregulatory loop with the progesterone receptor (PGR) and colocalizes with PGR on chromatin to promote PGR transcriptional programs. GATA2 also has PGR-independent functions that maintain endometrial cell identity, and GATA2 transcripts reportedly are down-regulated in endometrial disorders including endometriosis. This event is accompanied by a reciprocal increase in GATA6. Here, we applied custom anti-GATA2 monoclonal antibodies and performed GATA2 immunohistochemistry (IHC) on patient endometrial tissues corresponding to proliferative, secretory, inactive, and hormone-treated endometrium, as well as endometriosis and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN). We also performed IHC for the estrogen receptor, PGR, and GATA6 in relevant groups. The results reveal a tight correlation between GATA2 and PGR expression in the glandular and stromal cells of benign endometrium. GATA2 expression is markedly reduced in stromal but not glandular cells in endometriosis and EAH/EIN. This reduction in GATA2 expression does not lead to a detectable increase in GATA6 expression in endometriosis. Although average glandular GATA2 expression was preserved in endometriosis and EAH/EIN cases, its expression was decoupled from PGR, implying that alternative pathways regulate GATA2 levels in these disorders. Our findings indicate that GATA2 dysregulation is a feature of endometriosis and EAH/EIN, and support a model whereby loss of stromal GATA2 in these disorders contributes to their progesterone insensitivity.

Putrescine supplementation improves the developmental competence of in vitro produced bovine embryos

The aim of this study was to investigate the effect of putrescine, anti-apoptotic, antioxidant, and a cell proliferation stimulant, on embryo development and quality by supplementing it to in vitro culture medium. In this study, oocytes were obtained from the ovaries of Holstein cattle. Following maturation and fertilization, the presumptive zygotes were randomly assigned to two groups. The first group (Putrescine, n = 435) was supplemented with putrescine at a concentration of 0.5 mM to in vitro culture. The second group (n = 407) was maintained under standard culture conditions without any supplementations to the medium. Following the determination of the developmental stages of the embryos, only those in the blastocyst stage were subjected to differential staining and the cell numbers of the embryos were determined. Moreover, the TUNEL assay was employed to ascertain the extent of cell death and the apoptotic index in the embryos. Additionally, the levels of ROS were determined in the embryos. Furthermore, gene expression analyses were conducted on blastocyst-stage embryos to ascertain the potential of putrescine supplementation in embryo development along specific pathways. Following in vitro culture, the blastocyst formation rate was 44.37 % in the putrescine group and 32.97 % in the control group (P < 0.05). The counts of ICM (60.60 ± 15.79 vs 50.73 ± 16.74), TE (117.70 ± 23.67 vs 94.0 ± 22.46), and TCC (178.30 ± 26.15 vs 144.73 ± 26.86) were found to be statistically higher in blastocysts developing after putrescine supplementation compared to the control group. Furthermore, the number of apoptotic cells (7.69 ± 2.17 vs 9.96 ± 3.99) and the apoptotic index (5.07 % vs 8.01 %) were found to be lower in the putrescine group in comparison to the control group. Nevertheless, it was established that the ROS level in the control group was approximately two-fold higher than in the putrescine group (P < 0.05). The findings also revealed that putrescine up-regulated the gene expression of SOD, GPX4, CAT, BCL2, NANOG and GATA3 while simultaneously down-regulating the BAX expression level. In conclusion, the supplementation of putrescine to the culture medium during in vitro bovine embryo production was found to contribute to the improvement of embryo quality and early embryonic development.

Evaluation of the immunomodulatory activity of probiotics mixture and sulfasalazine against acetic acid-induced colitis in a murine model.

Currently, the use of probiotics to treat inflammatory bowel diseases (IBD) is widely accepted because of their gut microbiota modulation capabilities and anti-inflammatory potential. The aim of this study is to examine the immunomodulatory outcomes of probiotics and sulfasalazine in the acetic acid-induced colitis murine model. The animals were randomly assigned to one of the seven groups. Following the induction of colitis, Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12, and sulfasalazine (SASP) were orally administered for 10 days. Subsequently, the in vitro anti-inflammatory effect on TNF-α and IL-10 in the supernatants of cultured spleen cells was assessed via ELISAs. Relative mRNA expression of ZO-1, MLCK, iNOS, TNFR2, ROR-γt, GATA-3, T-bet, and Foxp3 was determined using quantitative reverse‑transcription polymerase chain reaction (qRT‑PCR). The SASP plus probiotic mixture was more effective in alleviating colitis symptoms, and reducing disease activity scores, and mucosal inflammation. qRT-PCR analysis revealed a significant reduction in T-bet and RORγt levels, while Foxp3 and GATA-3 levels increased in the colons of colitis mice. In addition, the selected strains substantially inhibited the release of inflammatory markers. Administration of LA-5 + BB-12 + SASP resulted in considerably higher inhibition of NO production and cell proliferation than in the other groups (p < 0.001). Treatment with LA-5 + BB-12 + SASP also reduced TNF-α-mediated apoptosis in intestinal epithelial cells (IECs). Survey results highlight that the combination regimen could be a promising strategy for IBD therapy, warranting further study of its clinical application and long-term benefits.

GATA1 transcription factor targets the gene expression of B19 virus in HEK293 cell line

Backgrond/Aim: B19 virus (B19V) is a single strand DNA virus that has specific tropism to erythroid progenitor cells (EPCs). The virus enters to the cells via P antigen and coreseptors and induces infection and cell apoptosis. GATA1 has a high expression in EPC and is a critical transcription factor for the cells development and differentiation. As human EPCs are the main target of the virus infection that have high expression of GATA-1 as the critical transcription factor, the aim of this study was to investigate the effect of GATA1 cotransfection with B19V genome on expression of the viral mRNAs in HEK293 as non-permissive cell line to the virus that had no mRNA expression of GATA-1. Methods: HEK293 cells were transfected with pHI0 plasmid containing B19V genome and the plasmid of GATA1 genome. The quantity of B19V mRNAs (NS1, 7.5 kDa, 11 kDa) expression was evaluated after 24h of transfection. Results: The results showed a statistically significant increase in fold change expression of (NS1 ∽ 12.3, VP1 ∽ 27.6, 11kb protein ∽ 38) in cotransfected cells with GATA1 and B19 plasmids compare to control group (P&lt;0.05). Conclusion: This research showed transfected cells with GATA1had elevation in expression of the B19V genes mRNAs in a non-permissive cell. This result may show a role of GATA1 as a critical transcription factor in support of the virus infection in EPCs. This suggests that GATA1 may potentially sport B19V replication or gene expression.

The protective effects and mechanisms of essential oil from Chimonanthus nitens Oliv. leaves in allergic rhinitis based on the NF-κB and T-bet/GATA-3 signaling pathways

Ethnopharmacological relevancePreliminary studies showed that Shanlameiye granules are derived from Chimonanthus nitens Oliv. Leaves ameliorate inflammatory responses in mice with Allergic Rhinitis (AR). The essential oil from Chimonanthus nitens Oliv. Leaves (CLO) have been identified as the key active substances in these granules. However, whether CLO constitutes the primary mechanism for the mitigation of AR-related inflammation by these granules has not yet been investigated. Aim of the studyThis experiment was to validate the effects and mechanism of CLO on inflammatory responses in RAW264.7 cells and AR rat model. Materials and methodsAn inflammatory model was induced in RAW264.7 cells by Lipopolysaccharide (LPS) & Interferon-gamma (IFN-γ) stimulation. AR rat model was established using both systemic and local challenges with Ovalbumin (OVA). ResultsIn cell experiments, CLO obviously decreased the secretion of cytokines and inhibited the NF-κB signaling pathway activation. In animal experiments, CLO decreased the number of eosinophils in the blood and lowered the levels of cytokines in nasal lavage fluid (NALF). Additionally, CLO inhibited the expression of STAT6, GATA-3, and p-p65, while increasing the expression of STAT4 and T-bet in the nasal mucosa. ConclusionIn AR rat model, CLO may play an anti-inflammatory role in AR rat model by regulating NF-κB and T-bet/GATA-3 signaling pathways.

DNA Methylation Negatively Regulates Gene Expression of Key Cytokines Secreted by BMMCs Recognizing FMDV-VLPs

Virus-like particles (VLPs) have been studied and used as vaccines to control foot-and-mouth disease (FMD). Mast cells (MCs) express various pattern recognition receptors that recognize pathogens and secrete numerous cytokines to initiate and modulate immune responses. Our previous study showed that bone marrow-derived mast cells (BMMCs) can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) to differentially express various cytokines and that histone acetylation can regulate the cytokines secreted during BMMC recognition of FMDV-VLPs. To demonstrate the role of DNA methylation in this response process, BMMCs that recognize FMDV-VLPs were treated with azacytidine (5-AZA), an inhibitor of DNA methylation transferase. We prepared FMDV-VLPs as described previously and cultured the BMMCs. The transcription and expression of key cytokines and transcription factors were determined using real-time quantitative PCR (RT-qPCR) and Western blotting. Results showed that pre-treatment with AZA resulted in the increased transcription and expression of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-13, and IL-10, while the changes in IL-13 transcription and IL-6 expression were irrelevant to mannose receptors (MRs). Furthermore, analysis of the transcription factors indicated that both the transcription and expression of nuclear factor-kappa B (NF-κB) increased significantly in the AZA pre-treated group, indicating that DNA methylation may also regulate NF-κB expression to modulate TNF-α, IL-13, and IL-6. However, pre-treatment with AZA did not alter the expression of microphthalmia-associated transcription factor (MITF) or GATA-2. All the data demonstrate that DNA methylation negatively regulates the transcription and expression of TNF-α, IL-13, IL-10, and IL-6 secreted by recognizing FMDV-VLPs. These results provide new ideas for the mast cell-based design of more effective vaccine adjuvants and targeted therapies in the future.

GATA2 downregulation contributes to pro-inflammatory phenotype and defective phagocytosis of pulmonary macrophages in chronic obstructive pulmonary disease.

Pulmonary macrophages from COPD patients are characterized by lower phagocytic and bactericidal activity whereas there is hypersecretion of pro-inflammatory cytokines. The prominent decline of GATA2 expression in pulmonary macrophages from COPD patients inspired us to figure out its role during COPD development. The expression levels of GATA2 were decreased in alveolar macrophages isolated from cigarette smoke (CS)-induced COPD mice and cigarette smoke extract (CSE)-treated macrophages. In vitro, both CSE and GATA2 knockdown via siRNAs elevated pro-inflammatory cytokines expression whereas inhibiting phagocytosis in macrophages. Integrated analysis of transcriptomics of GATA2-knockdown macrophages and the results of ChIP sequencing of GATA2 together with dual-luciferase reporter assay identified Abca1 and Pacsin1 as functional target genes of GATA2. Mechanistically, ABCA1 mediates the pro-inflammatory secretion phenotype and the dysfunction in early stage of phagocytosis of macrophages through TLR4/MyD88 and MEGF10/GULP1 pathways, respectively. PACSIN1/SUNJ1 partially mediates the disruption effects of GATA2 downregulation on maturation of phagolysosomes in macrophages. Together, our study suggests that GATA2 influences multiple functions of pulmonary macrophages by simultaneous transcriptional regulation of several target genes, contributing to the dysfunctions of pulmonary macrophages in response to CS, which provides an impetus for further investigations of GATA2 or other underappreciated transcription factors as regulatory hubs in COPD pathogenesis.

Evaluation of TNF-α and IFN-γ primed conditioned medium of mesenchymal stem cell in acetic acid-induced mouse model of acute colitis

IBD, an autoimmune-inflammatory disorder that affects people who are genetically prone to inflammation. There is a lot of interest in MSC-CM therapy, especially when primed with TNF-α + IFN-γ. Throughout the study, data were collected on the percentage of apoptotic cells, gene expression of ZO-1, Foxp3, GATA3, IDO-1, Muc2, T-bet, Notch1, TNFR2, and ROR-γt, colon weight and length, histopathological analysis, and DAI. TNF-α and IL-10 levels were assessed in addition to the NO level. The results suggest that primed MSC-CM improved DAI, mucosal deterioration, intestinal inflammation and NO concentration. The amount of TNF-α was decreased, but IL-10 and the colon’s percentage of apoptotic cells was increased. The mRNA expression of ZO-1, Foxp3, GATA3, IDO-1, and Muc2 genes increased greatly in the treatment groups, while the expression of T-bet, Notch1, TNFR2, and ROR-γt genes has decreased. These studies suggest that primed MSC-CM may combine with common treatments to improve responsiveness.

Growth hormone is involved in GATA1 gene expression via STAT5B in human erythroleukemia and monocytic cell lines

GATAs are a family of transcription factors consisting of six members. Particularly, GATA1 and GATA2 have been reported to promote the development of erythrocytes, megakaryocytes, eosinophils, and mast cells. However, little information is available on the extracellular ligands that promote GATA1 expression. We evaluated whether growth hormone (GH) is an extracellular stimulator that participates in the signal transduction of GATAs, focusing on GATA1 expression in hematopoietic cell lineages. We used a reporter assay, RT-PCR, real-time quantitative PCR, and western blotting to evaluate GH-induced expression of GATA1 and GATA2 in the human erythroleukemic cell line K562 and the non-erythroid cell line U937. GATA1 expression in these hematopoietic cell lines increased at the transcriptional and protein levels in the presence of GH, and was inhibited by a STAT5 specific inhibitor. Cells transfected with activated STAT5B showed increased expression of GATA1. We identified functional STAT5B consensus sequences as binding site-158 bp from the transcription starting site in the GATA1 promoter region. These results suggest that GH directly induces GATA1 expression via GHR/JAK/STAT5 and is related to hematopoietic cell proliferation.

Enoximone alleviates atopic dermatitis-like skin inflammation via inhibition of type 2 T helper cell development

Atopic dermatitis (AD) is an inflammatory skin disease that affects approximately 15–20 % of the children and 1–3 % of the adults worldwide. Corticosteroids and calcineurin inhibitors are used in AD therapy; however, they cause various side effects. Current studies focus on novel therapeutic targets such as phosphodiesterases (PDEs) to mitigate AD. However, the relationship between PDE3 inhibitors and AD has not yet been reported. This study aimed to investigate the therapeutic effects and pharmaceutical mechanisms of enoximone (Enox), a PDE3 inhibitor. Mice were stimulated with 2,4-dinitrochlorobenzene (DNCB) to induce AD-like skin inflammation and were topically treated with Enox for 2 weeks. Treatment with Enox reduced the dermatitis score, skin water loss, IgE production, and expression of cytokines and chemokines that were elevated by DNCB. Histologically, Enox treatment reduced the skin thickness and the infiltration of various inflammatory cells, including macrophages, mast cells, eosinophils, and type 2 T helper (Th2) cells. HuT78, a human T cell line, was used to investigate the differentiation of T cells into Th2 cells. Enox treatment decreased the expression of Th2 cytokines and GATA3, a Th2 cell marker in HuT78, and suppressed signaling pathways that play a crucial role in Th2 cell differentiation. Collectively, the results demonstrate that Enox alleviates AD-like skin inflammation by inhibiting T-cell development. Thus, Enox may be a therapeutic candidate for the treatment of AD.

Transcriptional Activity of Genes Regulating T-Helper Differentiation in the Accidentally Exposed Population of the Southern Urals.

The objective of this work was to study the expression of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes responsible for the regulation of the differentiation of various T-helper subpopulations in individuals chronically exposed to radiation. The object of the study was peripheral blood cells obtained from 120persons chronically exposed to radiation in a wide range of doses on the Techa River. The mean cumulative absorbed dose to red bone marrow in the examined exposed individuals was 742.7 ± 78.6 mGy (dose range, 73.5-3516.1 mGy); in the comparison group, 17.4 ± 2.2 mGy (dose range, 0.0-55.5 mGy). The subpopulation composition of T-helpers (Th1, Th2, and Th17) was analyzed by flow cytofluorometry. The relative mRNA content of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes was estimated by real-time PCR. The study made it possible to note a decrease in the relative number of T-helpers 2 in the populations of T-helpers of the central memory in the group of chronically exposed persons compared to the comparison group. In the population of T-helpers of the central memory, a statistically significant increase in the relative number of T-helpers 1 was shown, depending on the accumulated absorbed dose to red bone marrow. No changes in mRNA expression of the studied genes were observed. The analysis of the correlation between the expression of GATA3, MAPK8, STAT3, RORC, and TBX21 mRNA and the relative number of cells in subpopulations of T-helper types 1, 2, and 17 in the examined people did not reveal statistically significant patterns.

Abstract C070: Keratin 17 and GATA6 correlate with diffusely infiltrative versus gland-forming components of PDAC: Uncovering the transitional state in pancreatic ductal adenocarcinoma

Abstract Purpose: Optimization of pancreatic ductal adenocarcinoma (PDAC) patient survival will depend on advancing our understanding of the mechanisms governing tumor cell plasticity and effectively mitigating their invasive and metastatic capabilities. This study aims to investigate the correlation of cancer biomarkers crucial to these processes by integrating molecular signatures with histopathological characteristics using single-cell RNA sequencing (scRNA-seq) and multiplexed IHC/IF. Methods: Single-cell RNA sequencing (scRNA-seq) data from a comprehensive dataset of over 53,000 PDAC cells and validation of lead targets through 14-plex immunofluorescence, using a customized MICS platform (Miltenyi Biotec) and dual color IHC were employed to assess protein expression of defining biomarkers of basal and classical subtypes of PDAC including Keratin 17 (K17) and GATA6. Results: scRNAseq analysis revealed distinct patterns of K17 and GATA6 expression, highlighting intratumoral heterogeneity among PDAC tumors. Specifically, populations of cells expressing K17+/GATA6+ and K17+/GATA6- exhibited significant enrichment in pathways associated with invasion and cell migration, including PI3K/Akt/mTOR, TGF-β, and epithelial-mesenchymal transition (EMT) signaling. Conversely, GATA6-/K17- cells demonstrated enrichment in biosynthetic and metabolic pathways that govern cell cycle regulation. Additionally, mIF data assessment revealed correlations between histomorphological patterns and dynamic shifts in K17 and GATA6 expression within PDAC tumors. Diffusely infiltrative tumor cells (DITCs) uniformly expressed K17, contrasting with the gland-forming components (GFCs) of PDAC, which showed enrichment in GATA6 and sporadic expression of K17. E-cadherin, a key adhesion marker, exhibited the highest expression levels in the glandular and solid components of PDAC, underscoring their epithelial characteristics. Conversely, vimentin, a marker typically associated with epithelial-to-mesenchymal transition (EMT), was significantly expressed in DITCs, suggesting a mesenchymal-like phenotype in these infiltrative cells. Dual color IHC further validated these findings, reinforcing the correlation between morphologic plasticity and the expression patterns of K17 and GATA6, emphasizing their potential to distinguish distinct tumor cell populations within PDAC. Conclusions: These insights suggest that the interaction between these markers may indicate a distinct biological state characterized by altered cellular metabolism and potentially different therapeutic vulnerabilities. The robust association between K17 expression and diffusely infiltrative tumor cells underscores its potential as a marker for the most aggressive forms of this disease. Our results highlight the heterogeneous nature of PDAC and emphasize the critical importance of understanding these molecular distinctions in histopathological contexts to develop targeted therapies aimed at improving patient survival. Citation Format: Lyanne A Delgado- Coka, Brian Nelson, Michael Horowitz, Shayan Sarkar, Natalia Marchenko, Scott Powers, Luisa F Escobar-Hoyos, Kenneth Shroyer. Keratin 17 and GATA6 correlate with diffusely infiltrative versus gland-forming components of PDAC: Uncovering the transitional state in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C070.

Abstract B090: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths, with a 5-year survival rate of just 13%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non- Hispanic Black (defined here as African and/or African American ancestry) patients compared to other races. To identify molecular features that may contribute to racial health disparities in PDAC, we used quantitative mass spectrometry to determine the proteomic signatures unique to tumor racial origin in a cohort of 30 Caucasian and 12 African American patients. This analysis of the bulk tumor proteome revealed 183 proteins were differentially expressed between these groups. The protein with the highest expression in tumors from Black patients relative to Whites was Ring Finger Protein 2 (RNF2) that acts as an epigenetic transcriptional repressor in developmental processes pertaining to cellular differentiation. To determine the molecular pathways in PDAC regulated by RNF2, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 and a primary PDAC cell line derived from a Black patient to identify binding sites for RNF2 in PDAC cells. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6, which is an important determinant in PDAC subtype delineation with higher GATA6 expression associated with the less aggressive and more chemosensitive Classical subtype. Modulating RNF2 levels/activity by exogenous expression, short hairpin RNA (shRNA) knockdown, and small molecule inhibition confirms that GATA6 is a target of RNF2 repression in PDAC. Multi-omic (transcriptomic, proteomic, and phosphoproteomic) characterization was performed on MIA-PaCa2 cells with CRISPR-Cas9 knockout (KO) of RNF2. Differential gene expression (DE) analysis revealed changes in platinum drug resistance, EMT, and innate immune response, which are characteristic of the aggressive Inflammatory PDAC subtype we previously reported using proteomics. Total and phospho-proteomics corroborated these results identifying changes in DNA repair, inflammation response, TNFα mediated signaling, and apoptotic signaling. These results reflect the enrichment of Inflammatory subtype markers in tumors originating in Black patients. In agreement with these results, RNF2 KO shows a significant reduction in migration, as well as an increase in sensitivity to oxaliplatin. Our work identifies differences in the underlying proteome of PDAC tumors associated with tumor racial origin and describes how elevated expression of RNF2 in PDACs from Black patients contribute to inflammation and subtype delineation. This study delineates the associations between tumor racial origin, subtype specification, and response to therapy that could mitigate PDAC health disparities and be broadly applied across all PDAC patients. Citation Format: Anthony E Johansen-Sallee, Alexa M Barber, Henry C.-H. Law, Vignesh Vudatha, Jose Trevino, Nicholas T Woods. Evaluating the molecular determinants of PDAC racial health disparities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B090.

Expression of Transcriptional Factors of T Helper Differentiation (T-bet, GATA-3, RORγt, and FOXP3), MIF Receptors (CD44, CD74, CXCR2, 4, 7), and Th1, Th2, and Th17 Cytokines in PBMC from Control Subjects and Rheumatoid Arthritis Patients.

The macrophage migration inhibitory factor (MIF) plays a pivotal role in the development of rheumatoid arthritis (RA). Previous research indicates that MIF can trigger the expression of cytokine profiles associated with Th1, Th2, and Th17 responses in peripheral blood mononuclear cells (PBMC) from both RA patients and control subjects (CS). Despite these, few studies to date precisely elucidate the molecular mechanisms involved. The present study aimed to associate the expression of Th differentiation TF (T-bet, GATA-3, RORγt) with MIF receptors (CD44, CD74, CXCR2, 4, 7) and Th1, Th2, and Th17 cytokines in PBMC from CS and RA patients. PBMC from both groups was cultured for 24 h. The expression of the canonical and non-canonical MIF receptors and the TF was determined by flow cytometry. Additionally, multiplex bead analysis was employed to assess the levels of cytokines in the culture supernatants. The findings revealed that T CD4+ lymphocytes in the CS group exhibited a heightened expression of CD74 (p<0.05), whereas RA patients displayed an elevated expression of CXCR7 (p<0.001). Furthermore, T CD4+ lymphocytes from RA patients exhibited greater expression of GATA3, RORγt, and FOXP3, along with elevated levels of pro-inflammatory cytokines compared to the CS group (p<0.001). These results indicate that CD74 is more prominently expressed in PBMC from the CS group, whereas CXCR7 is more expressed in PBMC from RA patients. We also noted an increased secretion of Th17 profile cytokines in RA, potentially influenced by the activation of FOXP3 via CD74 and RORγt through CXCR7 using the endocytic pathway.

Immunohistochemical expression of GATA3, CK5/6 and CK20 in molecular subtypes of bladder carcinoma: correlation with clinicopathological features

BackgroundBladder urothelial carcinoma, is considered the 7th most common cancer in males. It is classified into luminal and basal subtypes depending on molecular markers, influencing prognosis and treatment. Identifying reliable biomarkers like GATA3, CK20, and CK5/6 through immunohistochemical methods can aid in early detection, risk stratification, and personalized treatment strategies. This study aims for evaluation prognostic role of these mentioned markers in correlation with clinicopathological parameters in urothelial carcinomas.MethodsTumor samples of forty cases were immunohistochemically stained for GATA3, CK5/6, and CK20. A cutoff of 20% positivity was used to determine subtype classifications, with staining patterns guiding the categorization into basal, luminal, double positive, or double negative groups.ResultsIn this study of 40 urothelial carcinoma patients tumors were classified into basal and luminal subtypes using GATA3, CK5/6 and CK20 markers. GATA3 expression showed no significant association with clinicopathological parameters; while, CK20 was associated with tumor size, and CK5/6 with T, N classification, and lymphovascular invasion. Significant differences in clinicopathological parameters were observed when subtypes were defined by CK5/6, GATA3 or CK20, particularly in tumor grade, T and N classification, and gender. Basal molecular subtypes was correlated with poor prognostic parameters.ConclusionsThis study documented that use of triple markers could define the luminal and basal subtypes of urothelial carcinoma. Basal tumors have shown to be associated with the aggressive behavior and future studies may allow the development of new therapies in the context of molecular subtypes.

Transcriptome-based classification to predict FOLFIRINOX response in a real-world metastatic pancreatic cancer cohort

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at metastatic stage and typically treated with fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX). Few patients benefit from this treatment. Molecular subtypes are prognostic in particularly resectable PDAC and might predict treatment response. This study aims to correlate molecular subtypes in metastatic PDAC with FOLFIRINOX responses using real-world data, providing assistance in counselling patients. We collected 131 RNA-sequenced metastatic biopsies and applied a network-based meta-analysis using published PDAC classifiers. Subsequent survival analysis was performed using the most suitable classifier. For validation, we developed an immunohistochemistry (IHC) classifier using GATA6 and keratin-17 (KRT17), and applied it to 86 formalin-fixed paraffin-embedded samples of advanced PDAC. Lastly, GATA6 knockdown models were generated in PDAC organoids and cell lines. We showed that the PurIST classifier was the most suitable classifier. With this classifier, classical tumors had longer PFS and OS than basal-like tumors (PFS: 216 vs. 78 days, p = 0.0002; OS: 251 vs. 195 days, p = 0.049). The validation cohort showed a similar trend. Importantly, IHC GATA6low patients had significantly shorter survival with FOLFIRINOX (323 vs. 746 days, p = 0.006), but no difference in non-treated patients (61 vs. 54 days, p = 0.925). This suggests that GATA6 H-score predicts therapy response. GATA6 knockdown models did not lead to increased FOLFIRINOX responsiveness. These data suggest a predictive role for subtyping (transcriptomic and GATA6 IHC), though no direct causal relationship was found between GATA6 expression and chemoresistance. GATA6 immunohistochemistry should be seamlessly added to current diagnostics and integrated into upcoming clinical trials.

Hypericum perforatum Alleviates Ovalbumin-Induced Asthma through Downregulating TH2 and Upregulating TH1 Related Parameters in BALB/C Mice

Background: Allergic asthma is a chronic and inflammatory disease of the respiratory tract, which is characterized by inflammation, airway obstruction and swelling. Anti-inflammatory compounds can be used to improve the disease severity. Hypericum perforatum (HP)/ St. John’s wort has been used in the treatment of many diseases due to its anti-inflammatory effects. Therefore, this study was performed to investigate the effects of HP on TH1 and TH2 parameters in an animal model of allergic asthma. Methods: Thirty BALB/c mice were sensitized with subcutaneous injection and inhalation of ovalbumin and then administered different doses of HP post-sensitization. Histological analysis of the lungs was performed. The effects of HP on mRNA expression of T-bet and GATA3 (TH1 and TH2 transcription factors, respectively), IFN-γ, IL-4 and IL-10 in the spleen were determined by real-time PCR. The protein levels of IFN-γ, IL-4 and IL-10 cytokines were also determined by ELISA technique in the serum of mice. Results: HP extract (HPE) reduced the clinical severity in asthma-induced mice, and a reduction in the inflammatory cell infiltration in the lung. Furthermore, treatment with HPE inhibited asthma-related mediators as well as increased the anti-inflammatory parameters compared to the control. The study also showed that treatment with HPE significantly increased IFN-γ, IL-10 and T-bet, while decreasing the level of IL-4 and GATA3 in mice treated with HPE. Conclusion: The data indicated that HPE can attenuate allergic asthma autoimmune responses by inhibiting infiltration of immune cell in the lung and TH2-related parameters. It is reasonable to assume that HPE has significant effects in the treatment of allergic asthma.