Breast cancer expression profiling has been used for determining biomarkers. Using gene expression profiles of 2,400 patients we identified keratin 19 (KRT19) as a highly deregulated gene in breast cancer. KRT19 expression is independent of patient race but correlates with disease grade, and ER, PR or HER2 expression. Expression of TPD52, GATA3 and KRT18 was increased in KRT19 expressing patients. Furthermore, KRT19 expression was associated with ER up-regulation and Luminal B gene signatures, as well as a constitutive RAF1 signaling pathway. Finally, KRT19 expression correlated with poor overall survival. Taken together, our results suggest that KRT19 expression can be used as a prognostic marker.

Allergic airway inflammation (AAI) in response to environmental antigens is an increasing medical problem, especially in the Western world. Type 2 interleukins (IL) are central in the pathological response but their importance and cellular source(s) often rely on the particular allergen. Here, we highlight the cellular sources and regulation of the prototypic type 2 cytokine, IL-13, during the establishment of AAI in a fungal infection model using Cryptococcus neoformans. IL-13 reporter mice revealed a rapid onset of IL-13 competence within innate lymphoid cells type 2 (ILC2) and IL-33R(+) T helper (Th) cells. ILC2 showed IL-33-dependent proliferation upon infection and significant IL-13 production. Th cells essentially required IL-33 to become either GATA3(+) or GATA3(+)/Foxp3(+) hybrids. GATA3(+) Th cells almost exclusively contributed to IL-13 production but hybrid GATA3(+)/Foxp3(+) Th cells did not. In addition, alveolar macrophages upregulated the IL-33R and subsequently acquired a phenotype of alternative activation (Ym1(+), FIZZ1(+), and arginase-1(+)) linked to type 2 immunity. Absence of adaptive immunity in rag2(-/-) mice resulted in attenuated AAI, revealing the need for Th2 cells for full AAI development. Taken together, in pulmonary cryptococcosis ILC2 and GATA3(+) Th2 cells produce early IL-13 largely IL-33R-dependent, thereby promoting goblet cell metaplasia, pulmonary eosinophilia, and alternative activation of alveolar macrophages.

Long non-coding RNAs (lncRNAs) have been identified in cerebral ischemia-reperfusion (I/R) injury nowadays. Herein, we uncovered the function and underlying mechanism of the lncRNA Rian in cerebral I/R injury. The oxygen-glucose deprivation model in N2a cells was offered to mimic cerebral I/R injury in vitro. Trypan blue staining, reactive oxygen species (ROS) production, and caspase-3 activity were used to evaluate cell apoptosis. Then, middle cerebral artery occlusion was conducted to evaluate the function of lncRNA Rian in mice. Real-time PCR and western blotting were performed to determine the expression of lncRNA Rian, miR-144-3p, GATA binding protein 3 (GATA3), caspase-3, Bax, and Bcl-2. The results showed that both Rian and GATA3 were downregulated, and miR-144-3p was upregulated in cerebral I/R injury in vitro and in vivo. Overexpression of Rian could inhibit the cell apoptosis induced by oxygen-glucose deprivation. Furthermore, overexpression of Rian distinctly reduced the infarct size, and it also improved the neurological score. Overexpression of Rian could abolish miR-144-3p-mediated I/R injury in vitro and in vivo. Besides, GATA3 was the target of miR-144-3p and GATA3 could be regulated co-operatively by miR-144-3p and Rian. Consequently, these findings showed that the Rian/miR-144-3p/GATA3 axis is an essential signaling in cerebral I/R injury. The lncRNA Rian may serve as a potential target for novel treatment in patients with ischemic stroke.

Delta-like ligand 4 (Dll4), one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti-VEGF therapy. Here, we examined the biologic effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biologic effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibit tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse-phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior antitumor effects in ovarian cancer models compared with either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel (P < 0.05 for the combination vs. aflibercept). The superior antitumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared with the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach. Mol Cancer Ther; 15(6); 1344-52. ©2016 AACR.

Inorganic arsenic has been claimed to increase the risk of pulmonary diseases through ingestion, as opposed to inhalation, which makes it a unique and intriguing environmental toxicant. However, the immunotoxic effects of lung, one of the targets of arsenic exposure, have not been extensively investigated in vivo. In the present study, we first confirmed that 2.5, 5 and 10mg/kg NaAsO2 orally for 24h dose-dependently triggered the infiltration of neutrophils, lymphocytes and macrophages in BALF. Not only the transcription activity, but also the secretion of proinflammatory cytokines IL-1β, IL-6 and TNF-α were consistently raised in the lung and BALF of acute arsenic-exposed mice. Acute oral administration of NaAsO2 also raised pulmonary MPO activity and mRNA levels of chemokine Mip-2 and Mcp-1. Meanwhile, obvious histopathological damages with inflammatory cells infiltration and erythrocyte aggregation around the capillaries were verified in the lung of mice drank arsenic-rich water freely for 3 months. Furthermore, we affirmed notable disturbance of CD4+ T-cell differentiation in the lung of acute arsenic-exposed mice, as demonstrated by up-regulated mRNA levels of regulator Gata3 and cytokine Il-4 of Th2, enhanced Foxp3 and Il-10 of Treg, down-regulated T-bet and Ifn-γ of Th1, as well as lessened Ror-γt and Il-23 of Th17. However, impressive elevation of cytokine Ifn-γ and Il-23, as well as moderate enhancement of Il-4 and Il-10 were found in the lung by subchronic arsenic administration. Finally, our present study demonstrated that both a single and sustained arsenic exposure prominently increased the expression of immune-related p38, JNK, ERK1/2 and NF-κB proteins in the lung tissue. While disrupting the pulmonary redox homeostasis by increasing MDA levels, exhausting GSH and impaired enzyme activities of CAT and GSH-Px, antioxidant regulator NRF2 and its downstream targets HO-1 and GSTO1/2 were also up-regulated by both acute and subchronic arsenic treatment. Conclusively, our present study demonstrated both acute and subchronic oral administration of arsenic triggers multiple pulmonary immune responses involving inflammatory molecules and T-cell differentiation, which might be closely associated with the imbalanced redox status and activation of immune-related MAPKs, NF-κB and anti-inflammatory NRF2 pathways.

BACKGROUND

Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Recent reports have shown that probiotics can induce immunomodulatory activity with promising effects in inflammatory diseases. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of Lactobacillus plantarum A7, which comprises human commensal bacteria, and Bifidobacterium animalis, a potential probiotic strain, on alleviation of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

METHODS

To evaluate the therapeutic effects of probiotic strains, female C57BL/6 mice (8-10 wks old) received Lactobacillus plantarum A7, Bifidobacterium animalis PTCC 1631or a mixture of both strains through oral administration daily for 22days beginning simultaneous with induction of EAE. The clinical parameters were recorded daily. On Day 22, each mouse was bled, and their spinal cord was removed for histology analysis. The effects of the treatments on regulatory T (Treg) cells level were evaluated using flow cytometry, and T-cell proliferation was assessed using a BrdU incorporation assay. The supernatants of spleen and lymph nodes cultured and mononuclear cells were collected for quantification of different panel of pro and anti-inflammatory cytokines by ELISA. The analysis of gene expression was performed at RNA level for transcription factors by real-time PCR.

RESULTS

The results showed that treatment with a mixture of the two strains caused a more significant delay in the time of disease onset and clinical score compared to when the strains were used alone. The pathological features of the disease, such as mononuclear infiltration into the CNS, were also inhibited more significantly by the combinational approach. The results also revealed that treatment with combination of both strains enhanced the population of CD4+CD25+Foxp3+-expressing T-cells in the lymph nodes and the spleen.

METHODS

with our probiotic strains markedly inhibited disease associated cytokines while increased anti-inflammatory cytokines. Additionally, L. plantarumA7 and B. animalis ameliorated EAE condition by favoring Th2 and Treg differentiation via up-regulation of Foxp3 and GATA3 in the brain and spleen as well as inhibited the differentiation of Th1 and Th17 cells.

CONCLUSIONS

The current research provided evidence that probiotic therapy with L. plantarum and B. animalis can effectively attenuate EAE progression as well as reinforce the polarization of regulatory T-cells.

Distinction between 2 forms of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), can be challenging. Aberrant mucosal immunity suggests that CD is a T helper type 1 cell (Th1)-driven disease, whereas UC as Th2-driven response. However, whether this paradigm truly distinguishes CD from UC is controversial. We aimed to clarify the discriminating potential of lamina propria Th subsets in patients with IBD.

Biopsies from 79 patients with IBD and 20 healthy controls were collected for Th subsets analysis (Th1:interferon γ [IFN-γ], T-bet; Th2:interleukin 13 [IL-13], Gata3; Th17:IL-17, RORγt; Treg:FoxP3). The receiver-operating characteristic curves were constructed to assess the discriminating ability by calculating the area under the receiver-operating characteristic curve. The equation with the highest area under the receiver-operating characteristic curve was applied to newly diagnosed patients to evaluate discriminating ability.

Patients with CD showed increased IFN-γ or T-bet cells and decreased IL-13 or Gata3 cells compared with UC. A discriminant equation composed of 4 markers (IFN-γ, T-bet, IL-13, and Gata3) yielded the highest area under the receiver-operating characteristic curve. In 36 established CD or UC, the sensitivity, specificity, positive and negative predictive probabilities were 92.6%, 55.6%, 86.2%, and 71.4% and in 14 newly diagnosed patients were 100.0%, 42.9%, 63.6%, and 100.0%. Furthermore, Gata3 cells were increased in tumor necrosis factor inhibitor therapy nonresponders compared with responders in CD. IFN-γ cells were directly and inversely proportional to disease activity in patients with CD and UC, respectively.

The Th1/Th2 paradigm can distinguish CD from UC and may be further associated with response to tumor necrosis factor inhibitor in CD and disease activity in patients with IBD.

Experienced T cells exhibit immunological memory via a rapid recall response, responding to restimulation much faster than naïve T cells. The formation of immunological memory starts during an initial slow response, when naïve T cells become transformed to proliferating T blast cells, and inducible immune response genes are reprogrammed as active chromatin domains. We demonstrated that these active domains are supported by thousands of priming elements which cooperate with inducible transcriptional enhancers to enable efficient responses to stimuli. At the conclusion of this response, a small proportion of these cells return to the quiescent state as long-term memory T cells. We proposed that priming elements can be established in a hit-and-run process dependent on the inducible factor AP-1, but then maintained by the constitutive factors RUNX1 and ETS-1. This priming mechanism may also function to render genes receptive to additional differentiation-inducing factors such as GATA3 and TBX21 that are encountered under polarizing conditions. The proliferation of recently activated T cells and the maintenance of immunological memory in quiescent memory T cells are also dependent on various cytokine signaling pathways upstream of AP-1. We suggest that immunological memory is established by T cell receptor signaling, but maintained by cytokine signaling.

BACKGROUND

The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.

METHODS

Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).

RESULTS

GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.

CONCLUSIONS

We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.

OBJECTIVE

To report a series of uterine corpus (n = 7) and ovarian (n = 5) neoplasms which we believe probably represent mesonephric adenocarcinomas based on their characteristic morphology and immunophenotype.

RESULTS

All neoplasms exhibited a relatively constant and characteristic morphological appearance with an admixture of architectural patterns with small glands or tubules, some containing luminal eosinophilic colloid-like material, typically predominating. Solid and papillary architectures were also often present. The nuclear features were characteristic with atypical angulated clear vesicular nuclei which often exhibited overlapping. All the tumours were 'flat' negative with oestrogen receptor and progesterone receptor and all except one exhibited nuclear staining with thyroid transcription factor 1 (TTF1), which was often diffuse. All tumours exhibited wild-type staining with p53. CD10, calretinin and GATA binding protein 3 (GATA3) were positive in a variable proportion of the neoplasms.

CONCLUSIONS

We believe these neoplasms to represent mesonephric adenocarcinomas which have only rarely been reported to arise in the uterine corpus and never in the ovary. We recommend they be termed mesonephric-like adenocarcinomas until their histogenesis is firmly established.

Mesonephric carcinomas of the gynecologic tract are neoplasms that are often under-recognized due to their varied morphologic appearances. Recently, GATA3 and TTF1 have been reported to be useful immunohistochemical markers for distinguishing mesonephric carcinomas from its morphologic mimics. Herein, we compared the performance of GATA3 and TTF1 to the traditional markers used for mesonephric carcinomas, CD10 and calretinin. We studied 694 cases: 8 mesonephric carcinomas (7 cervical [includes 3 mesonephric carcinosarcomas], 1 vaginal), 5 mesonephric-like carcinomas (4 uterine corpus, 1 ovarian), 585 endometrial adenocarcinomas, and 96 cervical adenocarcinomas. Mesonephric-like carcinomas were defined as tumors exhibiting the classic morphologic features of mesonephric carcinoma, but occurring outside of the cervix and without convincing mesonephric remnants. GATA3 had the highest sensitivity and specificity (91% and 94%) compared with TTF1 (45% and 99%), CD10 (73% and 83%), and calretinin (36% and 89%). GATA3, however, also stained a substantial number of uterine carcinosarcomas (23/113, 20%). TTF1 was positive in 5/5 (100%) mesonephric-like carcinomas and only 1/8 (13%) mesonephric carcinomas. In 4/6 (67%) TTF1 positive cases, GATA3 exhibited an inverse staining pattern with TTF1. In summary, GATA3 was the best overall marker for mesonephric and mesonephric-like carcinomas, but cannot be used to distinguish mesonephric carcinosarcomas from Müllerian carcinosarcomas. The inverse staining pattern between GATA3 and TTF1, suggests that TTF1 may be useful when GATA3 is negative in small biopsies where mesonephric or mesonephric-like carcinoma is suspected. The greater TTF1 positivity in mesonephric-like carcinomas suggests they may be biologically different from prototypical mesonephric carcinomas.

Bone marrow-derived monocyte-to-fibroblast transition is a key step in renal fibrosis pathogenesis, which is regulated by the inflammatory microenvironment. However, the mechanism by which the inflammatory microenvironment regulates this transition is not fully understood. In this study, we examined how the CD8(+) T cell/IFN-γ microenvironment regulates the monocyte-to-fibroblast transition in renal fibrosis. Genetic ablation of CD8 promoted a monocyte-to-fibroblast transition and increased renal interstitial fibrosis, whereas reconstitution of CD8 knockout (KO) mice with CD8(+) T cells decreased fibrosis. However, depletion of CD4(+) T cells in CD8 KO mice also reduced fibrosis. To elucidate the role of CD4(+) T cells in mediating CD8-regulated monocyte-to-fibroblast transition, CD4(+) T cells were isolated from obstructed kidneys of CD8 KO or wild-type mice. CD4(+) T cells isolated from CD8 KO obstructed kidney expressed more IL-4 and GATA3 and less IFN-γ and T-bet and showed increased monocyte-to-fibroblast transition in vitro compared with those isolated from wild-type obstructed kidney. To examine the role of IFN-γ-expressing CD8(+) T cells, we reconstituted CD8 KO mice with CD8(+) T cells isolated from IFN-γ KO mice. The IFN-γ KO CD8(+) cells had no effect on IL-4, GATA3, IFN-γ, and T-bet mRNA expression in obstructed kidneys or renal fibrosis. Taken together, our findings identify the axis of CD8(+) T cells and IFN-γ-CD4(+) T cells as an important microenvironment for the monocyte-to-fibroblast transition, which negatively regulates renal fibrosis.

Innate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional heterogeneity exists, and their differentiation pathways remain largely unclear. We applied mass cytometry to analyze ILCs in the human fetal intestine and distinguished 34 distinct clusters through a t-SNE-based analysis. A lineage (Lin)-CD7+CD127-CD45RO+CD56+ population clustered between the CD127+ ILC and natural killer (NK) cell subsets, and expressed diverse levels of Eomes, T-bet, GATA3, and RORγt. By visualizing the dynamics of the t-SNE computation, we identified smooth phenotypic transitions from cells within the Lin-CD7+CD127-CD45RO+CD56+ cluster to both the NK cells and CD127+ ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the Lin-CD7+CD127-CD45RO+CD56+CD8a- cells could develop into CD45RA+ NK cells and CD127+RORγt+ ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells.

This study was conducted to investigate the effect of morphine on CD4-positive T cells differentiation and the transcriptional factors induced by phorbol myristate acetate (PMA) and ionomycin. CD4-positive lymphocytes separated from healthy volunteers were incubated by PMA (25 ng/ml) + ionomycin (1 μg/ml) with or without the presence of morphine, ketamine, or naloxone. Th subsets, supernatant cytokines, and transcriptional factors were detected 4 h later. Th1 and Th2 cells, levels of INF-γ, IL-2, IL-4 and the activities of T-bet and GATA3 were significantly increased after incubation with PMA and ionomycin. However, the number of Th1 cells, Th1/ Th2, the levels of INF-γ and INF-γ/IL-4, and the activities and protein levels of T-bet and GATA3 were decreased after incubation with PMA and ionomycin in the presence of morphine. Naloxone can abolish morphine's suppressive effect on Th cell differentiation. Morphine has a negative effect on Th cell balance induced by PMA and ionomycin, the mechanism is related to T-bet and GATA3.

Glandular neoplasms involving the urinary bladder carry a challenging differential diagnosis including primary and secondary processes. We investigated the potential diagnostic utility of cadherin-17 and GATA3 in 25 primary adenocarcinomas of the urinary bladder, as compared with other commonly used markers including β-catenin and p63. Urothelial carcinoma with glandular differentiation (11), colorectal adenocarcinoma secondarily involving the bladder (25), and primary colorectal adenocarcinoma (22) were also analyzed and the results were compared using a Fisher exact test. Cadherin-17 was expressed in 23/25 primary bladder adenocarcinomas (92%), 23/25 colorectal adenocarcinomas involving the bladder (92%), 21/22 primary colorectal adenocarcinomas (95%) and entirely negative (0/11) in both components of urothelial carcinoma with glandular differentiation (P<0.001). In urothelial carcinoma with glandular differentiation, positive nuclear staining for GATA3 was evident in the urothelial component for 18% (2/11) and the glandular component for 9% (1/11) with additional tumors showing only cytoplasmic staining. Nuclear reactivity for GATA3 was not present in primary bladder adenocarcinoma and primary/secondary colorectal adenocarcinoma (P<0.05). Positive nuclear and cytoplasmic immunostaining for β-catenin was evident in 21/22 primary colorectal adenocarcinomas (95%) and 23/25 cases of secondary involvement by colorectal adenocarcinoma (92%). In contrast, positive membranous and cytoplasmic staining for β-catenin was observed in 23/25 primary bladder adenocarcinomas (92%) and 11/11 urothelial carcinomas with glandular differentiation (100%, P<0.001). p63 was expressed only in the urothelial component of urothelial carcinoma with glandular differentiation and not in the glandular component (P<0.001). In summary, cadherin-17 is a relatively specific and sensitive marker for primary adenocarcinoma of the urinary bladder, distinguishing it from urothelial carcinoma with glandular differentiation. However, it does not distinguish primary bladder adenocarcinoma from secondary involvement by colorectal adenocarcinoma. The pattern of reactivity for β-catenin remains the most useful marker for distinguishing these two tumors.

GATA3 is a zinc-finger transcription factor, which is expressed in various normal and neoplastic tissues. Amongst tumors, it labels urothelial carcinoma, collecting duct carcinoma of the kidney, breast carcinoma, lymphoma and, uncommonly, endometrial carcinoma. Few studies have investigated its positivity in various neoplasms that may mimic urothelial neoplasms. In this study, we evaluated GATA3 expression in urinary bladder paragangliomas, which may closely mimic urothelial carcinomas. We retrieved 12 cases of paragangliomas from the urinary bladder and 20 cases of paragangliomas from non-urologic sites using the Hopkins Pathology Data Base system. GATA3 was positive in 10 of the 12 (83%) urinary bladder paragangliomas studied on routine slide sections. Most (6/12) of the staining was diffusely strong (3+) staining, whereas the rest (4/12) that were positive showed mixed intensities (strong 3+ to moderate 2+). The 20 paragangliomas from other sites were constructed into tissue microarrays, wherein three cores from each tumor were taken. Fifteen out of 20 (75%) paragangliomas outside of the bladder were positive for GATA3 staining. Moderate (2+) or strong (3+) staining was seen in 13/20 (65%) of extravesical paragangliomas, ranging from 5 to 100% of the cell labeling (mean 59%, median 60%). In the remaining 7/20 (35%) cases, only weak (2/7) or negative (5/7) immunoreactivity for GATA3 was seen. An additional 15 cases of metastatic paraganglioma from various primary sites were retrieved with 12 of 15 (80%) metastatic paragangliomas staining positively for GATA3. Overall, for paragangliomas, regardless of site, 78.7% were positive for GATA3. Recognition of this finding will aid pathologists in preventing a misdiagnosis of a urothelial tumor based on GATA3 expression, which is critical given the differences in treatment, follow-up and prognosis between bladder paragangliomas and urothelial carcinoma.

Often the distinction of cutaneous apocrine carcinoma from metastatic mammary apocrine carcinoma to the skin can be a diagnostic dilemma because both tumors share similar histologic features and have overlapping immunohistochemical profile. We compared the expression of adipophilin, cytokeratin 5/6, p63, GATA3, mammaglobin, androgen receptor, estrogen receptor, progesterone receptor, and HER2 by immunohistochemistry in 14 cutaneous apocrine carcinomas (11 primary tumors, 3 metastases) and 26 primary apocrine carcinomas of the breast. Whereas focal adipophilin staining was seen in 36% (5/14) of cutaneous apocrine carcinoma, strong and diffuse adipophilin staining was seen in 88% (22/25) of mammary apocrine carcinoma (P = .0013). Differences in estrogen receptor and progesterone receptor expression were also statistically significant (P = .018 and .043). Androgen receptor was strongly positive in all cutaneous and mammary cases. Although there was no significant difference in the frequency of expression of cytokeratin 5/6, p63, HER2, GATA3, and mammaglobin in cutaneous apocrine carcinoma versus mammary apocrine carcinoma, strong and diffuse cytokeratin 5/6 and/or mammaglobin expression were seen only in cutaneous apocrine carcinoma. In conclusion, cutaneous apocrine carcinoma is likely adipophilin- ER+ PR+/- HER2- and can exhibit strong and diffuse cytokeratin 5/6 and/or mammaglobin expression. On the contrary, a mammary apocrine carcinoma is likely adipophilin+ ER- PR- and often exhibit 3+ HER2 with corresponding HER2 gene amplification. A panel of adipophilin, ER, PR, HER2, cytokeratin 5/6, and mammaglobin may be helpful in distinguishing cutaneous apocrine carcinoma from mammary apocrine carcinoma.

Kinases have been implicated in the immunopathological mechanisms of Systemic Lupus Erythematosus (SLE). v-akt murine-thymoma viral-oncogene-homolog 1 (AKT1) and mitogen-activated-protein-kinase 1 (MAPK1) gene expressions in peripheral mononuclear cells from thirteen SLE patients with inactive or mild disease were evaluated using quantitative real-time reverse-transcription polymerase-chain-reaction and analyzed whether there was any correlation with T-helper (Th) transcription factors (TF) gene expression, cytokines, and S100A8/S100A9-(Calprotectin). Age- and gender-matched thirteen healthy controls were examined. AKT1 and MAPK1 expressions were upregulated in SLE patients and correlated with Th17-(Retinoic acid-related orphan receptor (ROR)-C), T-regulatory-(Treg)-(Transforming Growth Factor Beta (TGFB)-2), and Th2-(interleukin (IL)-5)-related genes. MAPK1 expression correlated with Th1-(IL-12A, T-box TF-(T-bet)), Th2-(GATA binding protein-(GATA)-3), and IL-10 expressions. IL-10 expression was increased and correlated with plasma Tumor Necrosis Factor (TNF)-α and Th0-(IL-2), Th1-(IL-12A, T-bet), GATA3, Treg-(Forkhead/winged-helix transcription factor- (FOXP)-3), and IL-6 expressions. FOXP3 expression, FOXP3/RORC, and FOXP3/GATA3 expression ratios were increased. Plasma IL-1β, IL-12(p70), Interferon-(IFN)-γ, and IL-6 cytokines were augmented. Plasma IL-1β, IL-6, IL-2, IFN-γ, TNF-α, IL-10, and IL-13 correlated with C-reactive protein, respectively. Increased Calprotectin correlated with neutrophils. Conclusion, SLE patients presented a systemic immunoinflammatory activity, augmented AKT1 and MAPK1 expressions, proinflammatory cytokines, and Calprotectin, together with increased expression of Treg-related genes, suggesting a regulatory feedback opposing the inflammatory activity.

GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice.

We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice.

Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice.

Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.

Lung cancer is a leading cause of cancer-related mortality worldwide, and cigarette smoking is the major environmental hazard for its development. This study intended to examine whether smoking could alter methylation of genes at lung cancer risk loci identified by genome-wide association studies (GWASs). By systematic literature review, we selected 75 genomic candidate regions based on 120 single-nucleotide polymorphisms (SNPs). DNA methylation levels of 2854 corresponding cytosine-phosphate-guanine (CpG) candidates in whole blood samples were measured by the Illumina Infinium Human Methylation450 Beadchip array in two independent subsamples of the ESTHER study. After correction for multiple testing, we successfully confirmed associations with smoking for one previously identified CpG site within the KLF6 gene and identified 12 novel sites located in 7 genes: STK32A, TERT, MSH5, ACTA2, GATA3, VTI1A and CHRNA5 (FDR <0.05). Current smoking was linked to a 0.74% to 2.4% decrease of DNA methylation compared to never smoking in 11 loci, and all but one showed significant associations (FDR <0.05) with life-time cumulative smoking (pack-years). In conclusion, our study demonstrates the impact of tobacco smoking on DNA methylation of lung cancer related genes, which may indicate that lung cancer susceptibility genes might be regulated by methylation changes in response to smoking. Nevertheless, this mechanism warrants further exploration in future epigenetic and biomarker studies.

The spectrum of genomic alterations in ductal carcinoma in situ (DCIS) is relatively unexplored, but is likely to provide useful insights into its biology, its progression to invasive carcinoma and the risk of recurrence. DCIS (n=20) with a range of phenotypes was assessed by massively parallel sequencing for mutations and copy number alterations and variants validated by Sanger sequencing. PIK3CA mutations were identified in 11/20 (55%), TP53 mutations in 6/20 (30%), and GATA3 mutations in 9/20 (45%). Screening an additional 91 cases for GATA3 mutations identified a final frequency of 27% (30/111), with a high proportion of missense variants (8/30). TP53 mutations were exclusive to high grade DCIS and more frequent in PR-negative tumors compared with PR-positive tumors (P=0.037). TP53 mutant tumors also had a significantly higher fraction of the genome altered by copy number than wild-type tumors (P=0.005), including a significant positive association with amplification or gain of ERBB2 (P<0.05). The association between TP53 mutation and ERBB2 amplification was confirmed in a wider DCIS cohort using p53 immunohistochemistry as a surrogate marker for TP53 mutations (P=0.03). RUNX1 mutations and MAP2K4 copy number loss were novel findings in DCIS. Frequent copy number alterations included gains on 1q, 8q, 17q, and 20q and losses on 8p, 11q, 16q, and 17p. Patterns of genomic alterations observed in DCIS were similar to those previously reported for invasive breast cancers, with all DCIS having at least one bona fide breast cancer driver event. However, an increase in GATA3 mutations and fewer copy number changes were noted in DCIS compared with invasive carcinomas. The role of such alterations as prognostic and predictive biomarkers in DCIS is an avenue for further investigation.

GATA3 is implicated in mammary epithelial development and breast cancer progression and is an evolving immunohistochemical marker in breast cancer. Often associated with estrogen receptor (ER) signaling, GATA3 expression has been reported in ER-negative breast cancers, but systematic evaluation of GATA3 expression in a large set of triple-negative breast cancers (TNBC) is lacking. Given low sensitivities of mammaglobin (MGB) and GCDFP15 in metastatic TNBC, additional markers for site of origin identification would be useful in this context. We examined immunohistochemical expression of GATA3 in a large group of treatment-naive TNBC (n = 111) and ER-positive (n = 39) and HER2-positive (n = 31) breast cancers with commonly used antibody clones, HG3-31 (GATA3-H) and L50-823 (GATA3-L), and compared GATA3, MGB, and GCDFP15. Respectively, GATA3-L and GATA3-H were positive in 66% and 44% of TNBC (P = .002), 93% and 79% of ER-/HER2+ tumors (P = .596), and 100% of ER+/HER2- and ER+/HER2+ tumors (P = 1.00 each). GATA3-L was technically and diagnostically more sensitive than GATA3-H in TNBC and was technically more sensitive in other subtypes. MGB (26%) and GCDFP15 (16%) were less sensitive for TNBC than other subtypes (P < .001). Notably, 56% and 36% of MGB-/GCDFP15- TNBC were positive with GATA3-L and GATA3-H, respectively (P = .027). Seventy percent of TNBC were positive for GATA3-L, MGB, or GCDFP15 compared with 49% using GATA3-H in the panel. GATA3 is a diagnostically useful marker for TNBC and is more sensitive than MGB and GCDFP15 combined. GATA3-L is more sensitive for TNBC than GATA3-H, and an immunopanel of GATA3-L, MGB, and GCDFP15 provides optimal diagnostic sensitivity for TNBC.

As previously shown, higher levels of NOTCH1 and increased NF-kappaB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency (than expected by chance) of NF-kappaB-binding sites (BS), including potentially novel NF-kappaB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappaB, and other important TFs on more primitive HSC sets.