The long noncoding RNA Falcor regulates Foxa2 expression to maintain lung epithelial homeostasis and promote regeneration.

Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

FOXA2-initiated transcriptional activation of INHBA induced by methylmalonic acid promotes pancreatic neuroendocrine neoplasm progression.

Pancreatic neuroendocrine neoplasms (PanNENs) are a group of highly heterogeneous neoplasms originating from the endocrine islet cells of the pancreas with characteristic neuroendocrine differentiation, more than 60% of which represent metastases when diagnosis, causing major tumor-related death. Metabolic alterations have been recognized as one of the hallmarks of tumor metastasis, providing attractive therapeutic targets. However, little is known about the molecular mechanism of metabolic changes regulating PanNEN progression. In this study, we first identified methylmalonic acid (MMA) as an oncometabolite for PanNEN progression, based on serum metabolomics of metastatic PanNEN compared with non-metastatic PanNEN patients. One of the key findings was the potentially novel mechanism of epithelial-mesenchymal transition (EMT) triggered by MMA. Inhibin ßA (INHBA) was characterized as a key regulator of MMA-induced PanNEN progression according to transcriptomic analysis, which has been validated in vitro and in vivo. Mechanistically, INHBA was activated by FOXA2, a neuroendocrine (NE) specific transcription factor, which was initiated during MMA-induced progression. In addition, MMA-induced INHBA upregulation activated downstream MITF to regulate EMT-related genes in PanNEN cells. Collectively, these data suggest that activation of INHBA via FOXA2 promotes MITF-mediated EMT during MMA inducing PanNEN progression, which puts forward a novel therapeutic target for PanNENs.

Transcriptional network governing extraembryonic endoderm cell fate choice.

The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

A high concentrate diet inhibits forkhead box protein A2 expression, and induces oxidative stress, mitochondrial dysfunction and mitochondrial unfolded protein response in the liver of dairy cows.

High-concentrate diet induce subacute ruminal acidosis (SARA) and cause liver damage in ruminants. It has been reported that forkhead box protein A2 (FOXA2) can enhance mitochondrial membrane potential but its function in mitochondrial dysfunction induced by high concentrate diets is still unknown. Therefore, the aim of this study was to elucidate the effect of high-concentrate (HC) diet on hepatic FOXA2 expression, mitochondrial unfolded protein response (UPRmt), mitochondrial dysfunction and oxidative stress. A total of 12 healthy mid-lactation Holstein cows were selected and randomized into 2 groups: the low concentrate (LC) diet group (concentrate:forage = 4:6) and HC diet group (concentrate:forage = 6:4). The trial lasted 21 d. The rumen fluid, blood and liver tissue were collected at the end of the experiment. The results showed that the rumen fluid pH level was reduced in the HC group and the pH was lower than 5.6 for more than 4 h/d, indicating that feeding HC diets successfully induced SARA in dairy cows. Both FOXA2 mRNA and protein abundance were significantly reduced in the liver of the HC group compared with the LC group. The activity of antioxidant enzymes (CAT, G6PDH, T-SOD, Cu/Zn SOD, Mn SOD) and mtDNA copy number in the liver tissue of the HC group decreased, while the level of H2O2 significantly increased, this increase was accompanied by a decrease in oxidative phosphorylation (OXPHOS). The balance of mitochondrial division and fusion was disrupted in the HC group, as evidenced by the decreased mRNA level of OPA1, MFN1, and MFN2 and increased mRNA level of Drp1, Fis1, and MFF. At the same time, HC diet downregulated the expression level of SIRT1, SIRT3, PGC-1α, TFAM, and Nrf 1 to inhibit mitochondrial biogenesis. The HC group induced UPRmt in liver tissue by upregulating the mRNA and protein levels of CLPP, LONP1, CHOP, Hsp10, and Hsp60. In addition, HC diet could increase the protein abundance of Bax, CytoC, Caspase 3 and Cleaved-Caspase 3, while decrease the protein abundance of Bcl-2 and the Bcl-2/Bax ratio. Overall, our study suggests that the decreased expression of FOXA2 may be related to UPRmt, mitochondrial dysfunction, oxidative stress, and apoptosis in the liver of dairy cows fed a high concentrate diet.

FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

FoxA2 represses ERß-mediated pyroptosis in endometriosis by transcriptionally inhibiting IGF2BP1.

BACKGROUND: Endometriosis is a severe disease which is associated with excessive activation of pyroptosis. Our present research aimed to investigate the function of Forkhead Box A2 (FoxA2) in regulating pyroptosis in endometriosis. METHODS: IL-1ß and IL-18 concentrations were assessed using ELISA. Cell pyroptosis was analyzed using flow cytometry. TUNEL staining was performed to determine human endometrial stromal cells (HESC) death. Moreover, ERß mRNA stability was assessed using RNA degradation assay. Finally, the binding relationships between FoxA2, IGF2BP1 and ERß were verified by dual-luciferase reporter system, ChIP, RIP and RNA pull-down assays. RESULTS: Our results revealed that IGF2BP1 and ERß were significantly upregulated in ectopic endometrium (EC) tissues of endometriosis patients compared to that in eutopic endometrium (EU) tissues as well as IL-18 and IL-1ß levels. Loss-of-function experiments subsequently demonstrated that either IGF2BP1 knockdown or ERß knockdown could repress HESC pyroptosis. In addition, IGF2BP1 upregulation promoted the pyroptosis in endometriosis by binding to ERß and promoting ERß mRNA stability. Our further research displayed that FoxA2 upregulation suppressed HESC pyroptosis by interacting with IGF2BP1 promoter. CONCLUSION: Our research proved that FoxA2 upregulation downregulated ERß by transcriptionally inhibiting IGF2BP1, thereby repressing pyroptosis in endometriosis.

Study on the Mechanism of FOXA2 Activation on Glutathione Metabolic Reprogramming Mediated by ETV4 Transcription to Facilitate Colorectal Cancer Malignant Progression.

The metabolic imbalance of glutathione (GSH) has been widely recognized in most cancers, but the specific molecular mechanism of GSH metabolic regulation in the malignant progression of colorectal cancer (CRC) is unexplored. The objective of our project is to elucidate whether ETV4 affects the malignant progression of CRC through GSH metabolic reprogramming. Bioinformatics and molecular experiments measured the expression of ETV4 in CRC, and in vitro experiments explored the impact of ETV4 on CRC malignant progression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) identified the pathway of ETV4 enrichment. The bioinformatics approach identified FOXA2 as an upstream regulatory factor of ETV4. The dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) experiment verified the binding relationship between ETV4 and FOXA2. Cell viability, migration, and invasion abilities were determined by conducting CCK-8, wound healing, and Transwell assays, respectively. The expression levels of N-cadherin, E-cadherin, and vimentin were determined by utilizing immunofluorescence (IF). Metabolism-related enzymes GCLM, GCLC, and GSTP1 levels were detected to evaluate the GSH metabolism level by analyzing the GSH/GSSG ratio. In vivo experiments were performed to explore the effect of FOXA2/ETV4 on CRC progression, and the expression of related proteins was detected by western blot. ETV4 was highly expressed in CRC. Knocking down ETV4 suppressed CRC cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) progression in vitro. ETV4 was abundant in the GSH metabolic pathway, and overexpression of ETV4 facilitated CRC malignant progression through activation of the GSH metabolism. In addition, in vitro cellular experiments and in vivo experiments in nude mice confirmed that FOXA2 transcriptionally activated ETV4. Knocking down FOXA2 repressed the malignant phenotype of CRC cells by suppressing GSH metabolism. These effects were reversed by overexpressing ETV4. Our results indicated that FOXA2 transcriptionally activates ETV4 to facilitate CRC malignant progression by modulating the GSH metabolic pathway. Targeting the FOXA2/ETV4 axis or GSH metabolism may be an effective approach for CRC treatment.

FOXA2 suppresses gallbladder carcinoma cell migration, invasion, and epithelial-mesenchymal transition by targeting SERPINB5.

BACKGROUND: Gallbladder cancer (GBC), a highly malignant gastrointestinal tumor, lacks effective therapies. Foxhead box A2 (FOXA2) is a tumor suppressor that is poorly expressed in various human malignancies. This study aimed to ascertain FOXA2 expression in GBC and its relevance to tumor metastasis, and to elucidate its regulatory mechanism with epithelial-mesenchymal transition (EMT) as an entry point, in the hope of providing a potential therapeutic target for GBC. METHODS: FOXA2 expression in GBC tissues was first detected using immunohistochemistry (IHC), followed by correlation analysis with clinicopathological characteristics and survival prognosis. Subsequently, the effects of FOXA2 on GBC cell migration and invasion, as well as EMT induction, were evaluated by scratch, Transwell, RT-PCR, and Western blot assays, together with animal experimentation. Ultimately, mRNA sequencing was carried out to identify the key downstream target genes of FOXA2 in controlling the EMT process in GBC cells, and dual-luciferase reporter and chromatin immunoprecipitation assays were used to determine its regulatory mechanism. RESULTS: FOXA2 was underexpressed in GBC tissues and inversely correlated with tumor node metastasis stage, lymph node metastasis, and poor patient prognosis. FOXA2 exerts suppressive effects on EMT and metastasis of GBC in vivo and in vitro. FOXA2 can impede GBC cell migratory and invasive functions and EMT by positively mediating serine protein kinase inhibitor B5 (SERPINB5) expression. CONCLUSION: FOXA2 directly binds to the SERPINB5 promoter region to stimulate its transcription, thereby modulating the migration and invasion behaviors of GBC cells as well as the EMT process, which might be an effective therapeutic target against GBC.

FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner.

Differentiation of normal and tumor cells is controlled by regulatory networks enforced by lineage-determining transcription factors (TFs). Among them, TFs such as FOXA1/2 bind naïve chromatin and induce its accessibility, thus establishing new gene regulatory networks. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the coexistence of well- and poorly differentiated cells at all stages of disease. How the transcriptional networks determining such massive cellular heterogeneity are established remains to be determined. We found that FOXA2, a TF controlling pancreas specification, broadly contributed to the cis-regulatory networks of PDACs. Despite being expressed in both well- and poorly differentiated PDAC cells, FOXA2 displayed extensively different genomic distributions and controlled distinct gene expression programs. Grade-specific functions of FOXA2 depended on its partnership with TFs whose expression varied depending on the differentiation grade. These data suggest that FOXA2 contributes to the regulatory networks of heterogeneous PDAC cells via interactions with alternative partner TFs.

The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Updating germ cell tumour pathogenesis - the ability of seminomas for FOXA2-driven extra-embryonic differentiation.

AIMS: Testicular germ cell tumours are the most common solid malignancies in young men of age 14-44 years. It is generally accepted that both seminomas and non-seminomas arise from a common precursor, the germ cell neoplasia in-situ, which itself is the result of a defective (primordial) germ cell development. The stem cell-like population of the non-seminomas, the embryonal carcinoma, is capable of the differentiation of all three germ layers (teratomas) and extra-embryonic tissues (yolk-sac tumours, choriocarcioma) into cells. In contrast, seminomas are thought to have a limited differentiation potential. Nevertheless, several studies have highlighted their ability to undergo reprogramming to an embryonal carcinoma or differentiation into other non-seminomatous entities. Here, we demonstrate that in approximately 5% of seminomas, the yolk-sac tumour driver gene FOXA2 is detectable at the protein level, indicative of an occult yolk-sac tumour subpopulation that putatively arose from seminoma cells, as the presence of other GCT entities could be excluded. The presence of these subpopulations might render the tumour more aggressive and argue for an adjustment of the therapeutic concept. We used our data to update the model of germ cell tumour pathogenesis, especially regarding the developmental potential of seminomas. Additionally, we suggest to include detection of FOXA2 into standard routine diagnosis of seminomas.

FoxA2 is a reliable marker for the diagnosis of yolk sac tumour postpubertal-type.

AIMS: Yolk sac tumour postpubertal-type (YSTpt) shows a wide range of histological patterns and is challenging to diagnose. Recently, forkhead box transcription factor A2 (FoxA2) emerged as a driver of YSTpt formation and a promising marker for diagnosing YSTpt. However, FoxA2 has not been tested in the different patterns of YSTpt. This study aimed to assess the staining pattern of FoxA2 in te different patterns of YSTpt and other germ cell tumours of the testis (GCTT), comparing it with glypican-3 (GPC3) and α-fetoprotein (AFP). METHODS AND RESULTS: FOXA2, GPC3 and AFP immunohistochemistry was performed on 24 YSTpt (24 microcystic/reticular, 10 myxoid, two macrocystic, five glandular/alveolar, two endodermal sinus/perivascular, four solid, two polyembryoma/embryoid body and two polyvesicular vitelline) and 81 other GCTT. The percentage of positive cells (0, 1+, 2+, 3+) and the intensity (0, 1, 2, 3) were evaluated regardless of and within each YSTpt pattern. FoxA2 was positive in all YSTpt (24 of 24) and all but one (23 of 24) exhibited 2+/3+ stain, with higher intensity [median value (mv): 2.6] than AFP (1.8) and GPC3 (2.5). Both FoxA2 and GPC3 were positive in all microcystic/reticular (24 of 24), myxoid (10 of 10), macrocystic (two of two), endodermal sinus/perivascular (four of four) and polyembryoma/embryoid body (two of two) patterns. Nevertheless, only FoxA2 was positive in all glandular/alveolar (five of five), solid (four of four) and polyvesicular vitelline (two of two) patterns. The intensity of FoxA2 was higher than AFP and GPC3 in almost all YST patterns. In the other GCTT, FoxA2 was positive only in teratoma postpubertal-type (Tpt) [13 of 20 (65%)], with staining almost exclusively confined to the mature gastrointestinal/respiratory tract epithelium. CONCLUSIONS: FoxA2 is a highly sensitive and specific biomarker that supports the diagnosis of YSTpt. FoxA2 is superior to GPC3 and AFP, especially in rare and difficult-to-diagnose histological patterns of YSTpt, but mature glands of Tpt could represent a potential diagnostic pitfall.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2.

BACKGROUND: Hepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages. METHODS: Constitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6-/-) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells. RESULTS: We observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC. CONCLUSION: In summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

FOXA2 suppresses PKM2 transcription and affects the Wnt/ß-catenin activity to block aerobic glycolysis in thyroid carcinoma.

Aerobic glycolysis is critical for the energy metabolism of cancer cells. This study focuses on the regulation of forkhead box A2 (FOXA2) on pyruvate kinase M2 (PKM2) and their effects on the glycolytic activity and malignant phenotype of thyroid carcinoma (THCA) cells. By analysing four Gene Expression Omnibus datasets and querying bioinformatics systems, we obtained FOXA2 as a poorly expressed transcription factor in THCA. Later, we validated decreased mRNA and protein levels of FOXA2 in THCA cells by quantitative polymerase chain reaction and western blot assays. FOXA2 upregulation in THCA cells suppressed the glucose uptake and lactate production, and it reduced the extracellular acidification rate, but increased the oxygen consumption rate of cells. Meanwhile, the FOXA2 overexpression blocked the proliferation and mobility, and the tumourigenic activity of cancer cells. The chromatin immunoprecipitation and luciferase assays showed that FOXA2 bound to PKM2 promoter and suppressed the transcription of PKM2, which was highly expressed in THCA cells. Further upregulation of PKM2 elevated the ß-catenin, c-Myc and cyclin D1 levels and restored the glycolytic activity as well as the malignant properties of cancer cells. Collectively, this work reveals that FOXA2 suppresses aerobic glycolysis and progression of THCA by blocking PKM2 transcription and inactivating the Wnt/ß-catenin pathway.

Forkhead box A2-mediated lncRNA SOX2OT up-regulation alleviates oxidative stress and apoptosis of renal tubular epithelial cells by promoting SIRT1 expression in diabetic nephropathy.

BACKGROUND: Renal tubular injury is the main feature of diabetic nephropathy (DN). We intend to investigate the function and related mechanisms of lncRNA SOX2 overlapping transcript (SOX2OT) in high glucose (HG)-induced oxidative stress and apoptosis of renal tubular epithelial cells (RTECs). METHODS: To construct diabetes models, the human kidney-2 (HK-2) cells were treated with HG (30 mM), and mice were injected with streptozotocin. The levels of intracellular and mitochondrial reactive oxygen species (ROS) were assessed by dihydroethidium staining and MitoSox staining. The cell apoptosis was assessed by flow cytometry and TUNEL staining. Levels of serum creatinine, blood urea nitrogen (BUN), Urinary ACR, and oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were detected by relevant kits. In addition, fluorescence in situ hybridization staining, RNA-pull down, RNA immunoprecipitation (RIP), co-immunoprecipitation (co-IP), dual-luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) were also executed. RESULTS: Levels of SOX2OT and silent information regulator 1 (SIRT1) were down-regulated in HG-cultured HK-2 cells. Overexpressing SOX2OT reduced intracellular and mitochondrial ROS levels and cell apoptosis in vitro. Moreover, SOX2OT overexpression also reduced serum creatinine, BUN, urinary ACR, 8-OHdG, renal tubular injury markers KIM1 and NGAL, ROS levels, and cell apoptosis in vivo. In addition, SOX2OT promoted SIRT1 expression by suppressing its ubiquitination. Besides, interference with SIRT1 reversed the inhibitory effect of SOX2OT overexpression on HG-induced oxidative stress and apoptosis. Forkhead box A2 (Foxa2) levels were up-regulated in HG-cultured HK-2 cells. Foxa2 could bind to the SOX2OT promoter and suppress its expression. Furthermore, interfering with SOX2OT reversed the inhibitory effect of Foxa2 interference on HG-induced oxidative stress and apoptosis. CONCLUSION: Foxa2-mediated SOX2OT up-regulation reduced oxidative stress and apoptosis of RTECs by promoting SIRT1 expression, thus alleviating the progression of DN.

Sexually dimorphic effects of forkhead box a2 (FOXA2) and uterine glands on decidualization and fetoplacental development.

Glands of the uterus are essential for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands of the uterus and a critical regulator of glandular epithelium (GE) differentiation, development, and function. Mice with a conditional deletion of FOXA2 in the adult uterus, created using the lactotransferrin iCre (Ltf-iCre) model, have a morphologically normal uterus with glands, but lack FOXA2-dependent GE-expressed genes, such as leukemia inhibitory factor (LIF). Adult FOXA2 conditional knockout (cKO; LtfiCre/+Foxa2f/f ) mice are infertile due to defective embryo implantation arising from a lack of LIF, a critical implantation factor of uterine gland origin. However, intraperitoneal injections of LIF can initiate embryo implantation in the uterus of adult FOXA2 cKO mice with pregnancies maintained to term. Here, we tested the hypothesis that FOXA2-regulated genes in the uterine glands impact development of the decidua, placenta, and fetus. On gestational day 8.5, the antimesometrial and mesometrial decidua transcriptome was noticeably altered in LIF-replaced FOXA2 cKO mice. Viable fetuses were reduced in FOXA2 cKO mice on gestational days 12.5 and 17.5. Sex-dependent differences in fetal weight, placenta histoarchitecture, and the placenta and metrial gland transcriptome were observed between control and FOXA2 cKO mice. The transcriptome of the placenta with a female fetus was considerably more altered than the placenta with a male fetus in FOXA2 cKO dams. These studies reveal previously unrecognized sexually dimorphic effects of FOXA2 and uterine glands on fetoplacental development with potential impacts on offspring health into adulthood.

Subacute ruminal acidosis downregulates FOXA2, changes oxidative status, and induces autophagy in the livers of dairy cows fed a high-concentrate diet.

The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3ß (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.