HNF1B-driven three-dimensional chromatin structure for molecular classification in pancreatic cancers.

The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

Dysregulation of HNF1B/Clusterin axis enhances disease progression in a highly aggressive subset of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is largely refractory to available treatments. Identifying key pathways associated with disease aggressiveness and therapeutic resistance may characterize candidate targets to improve patient outcomes. We used a strategy of examining the tumors from a subset of PDAC patient cohorts with the worst survival to understand the underlying mechanisms of aggressive disease progression and to identify candidate molecular targets with potential therapeutic significance. Non-negative matrix factorization (NMF) clustering, using gene expression profile, revealed three patient subsets. A 142-gene signature specific to the subset with the worst patient survival, predicted prognosis and stratified patients with significantly different survival in the test and validation cohorts. Gene-network and pathway analysis of the 142-gene signature revealed dysregulation of Clusterin (CLU) in the most aggressive patient subset in our patient cohort. Hepatocyte nuclear factor 1 b (HNF1B) positively regulated CLU, and a lower expression of HNF1B and CLU was associated with poor patient survival. Mechanistic and functional analyses revealed that CLU inhibits proliferation, 3D spheroid growth, invasiveness and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cell lines. Mechanistically, CLU enhanced proteasomal degradation of EMT-regulator, ZEB1. In addition, orthotopic transplant of CLU-expressing pancreatic cancer cells reduced tumor growth in mice. Furthermore, CLU enhanced sensitivity of pancreatic cancer cells representing aggressive patient subset, to the chemotherapeutic drug gemcitabine. Taken together, HNF1B/CLU axis negatively regulates pancreatic cancer progression and may potentially be useful in designing novel strategies to attenuate disease progression in PDAC patients.

Mechanisms of ion transport regulation by HNF1ß in the kidney: beyond transcriptional regulation of channels and transporters.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor essential for the development and function of the kidney. Mutations in and deletions of HNF1ß cause autosomal dominant tubule interstitial kidney disease (ADTKD) subtype HNF1ß, which is characterized by renal cysts, diabetes, genital tract malformations, and neurodevelopmental disorders. Electrolyte disturbances including hypomagnesemia, hyperuricemia, and hypocalciuria are common in patients with ADTKD-HNF1ß. Traditionally, these electrolyte disturbances have been attributed to HNF1ß-mediated transcriptional regulation of gene networks involved in ion transport in the distal part of the nephron including FXYD2, CASR, KCNJ16, and FXR. In this review, we propose additional mechanisms that may contribute to the electrolyte disturbances observed in ADTKD-HNF1ß patients. Firstly, kidney development is severely affected in Hnf1b-deficient mice. HNF1ß is required for nephron segmentation, and the absence of the transcription factor results in rudimentary nephrons lacking mature proximal tubule, loop of Henle, and distal convoluted tubule cluster. In addition, HNF1ß is proposed to be important for apical-basolateral polarity and tight junction integrity in the kidney. Interestingly, cilia formation is unaffected by Hnf1b defects in several models, despite the HNF1ß-mediated transcriptional regulation of many ciliary genes. To what extent impaired nephron segmentation, apical-basolateral polarity, and cilia function contribute to electrolyte disturbances in HNF1ß patients remains elusive. Systematic phenotyping of Hnf1b mouse models and the development of patient-specific kidney organoid models will be essential to advance future HNF1ß research.

The mechanism and effects of remdesivir-induced developmental toxicity in zebrafish: Blood flow dysfunction and behavioral alterations.

The antiviral drug remdesivir has been used to treat the growing number of coronavirus disease 2019 (COVID-19) patients. However, the drug is mainly excreted through urine and feces and introduced into the environment to affect non-target organisms, including fish, which has raised concerns about potential ecotoxicological effects on aquatic organisms. Moreover, studies on the ecological impacts of remdesivir on aquatic environments have not been reported. Here, we aimed to explore the toxicological impacts of microinjection of remdesivir on zebrafish early embryonic development and larvae and the associated mechanism. We found that 100 µM remdesivir delayed epiboly and impaired convergent movement of embryos during gastrulation, and dose-dependent increases in mortality and malformation were observed in remdesivir-treated embryos. Moreover, 10-100 µM remdesivir decreased blood flow and swimming velocity and altered the behavior of larvae. In terms of molecular mechanisms, 80 differentially expressed genes (DEGs) were identified by transcriptome analysis in the remdesivir-treated group. Some of these DEGs, such as manf, kif3a, hnf1ba, rgn, prkcz, egr1, fosab, nr4a1, and ptgs2b, were mainly involved in early embryonic development, neuronal developmental disorders, vascular disease and the blood flow pathway. These data reveal that remdesivir can impair early embryonic development, blood flow and behavior of zebrafish embryos/larvae, probably due to alterations at the transcriptome level. This study suggests that it is important to avoid the discharge of remdesivir to aquatic ecosystems and provides a theoretical foundation to hinder remdesivir-induced ecotoxicity to aquatic environments.

Comprehensive quantitative analysis of alternative splicing variants reveals the HNF1B mRNA splicing pattern in various tumour and non-tumour tissues.

Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor and putative biomarker of solid tumours. Recently, we have revealed a variety of HNF1B mRNA alternative splicing variants (ASVs) with unknown, but potentially regulatory, functions. The aim of our work was to quantify the most common variants and compare their expression in tumour and non-tumour tissues of the large intestine, prostate, and kidney. The HNF1B mRNA variants 3p, Δ7, Δ7-8, and Δ8 were expressed across all the analysed tissues in 28.2-33.5%, 1.5-2%, 0.8-1.7%, and 2.3-6.9% of overall HNF1B mRNA expression, respectively, and occurred individually or in combination. The quantitative changes of ASVs between tumour and non-tumour tissue were observed for the large intestine (3p, Δ7-8), prostate (3p), and kidney samples (Δ7). Decreased expression of the overall HNF1B mRNA in the large intestine and prostate cancer samples compared with the corresponding non-tumour samples was observed (p = 0.019 and p = 0.047, respectively). The decreased mRNA expression correlated with decreased protein expression in large intestine carcinomas (p < 0.001). The qualitative and quantitative pattern of the ASVs studied by droplet digital PCR was confirmed by next-generation sequencing, which suggests the significance of the NGS approach for further massive evaluation of the splicing patterns in a variety of genes.

Hepatocyte nuclear factor-1ß shapes the energetic homeostasis of kidney tubule cells.

Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1ß (HNF-1ß) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1ß deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b-/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1ß in metabolism. Hnf1b-/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b-/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b-/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1ß. HNF-1ß shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.

Inhibition of hepatocyte nuclear factor 1ß contributes to cisplatin nephrotoxicity via regulation of nf-κb pathway.

Cisplatin nephrotoxicity has been considered as serious side effect caused by cisplatin-based chemotherapy. Recent evidence indicates that renal tubular cell apoptosis and inflammation contribute to the progression of cisplatin-induced acute kidney injury (AKI). Hepatocyte nuclear factor 1ß (HNF1ß) has been reported to regulate the development of kidney cystogenesis, diabetic nephrotoxicity, etc However, the regulatory mechanism of HNF1ß in cisplatin nephrotoxicity is largely unknown. In the present study, we examined the effects of HNF1ß deficiency on the development of cisplatin-induced AKI in vitro and in vivo. HNF1ß down-regulation exacerbated cisplatin-induced RPTC apoptosis by indirectly inducing NF-κB p65 phosphorylation and nuclear translocation. HNF1ß knockdown C57BL/6 mice were constructed by injecting intravenously with HNF1ß-interfering shRNA and PEI. The HNF1ß scramble and knockdown mice were treated with 30 mg/kg cisplatin for 3 days to induce acute kidney injury. Cisplatin treatment caused increased caspase 3 cleavage and p65 phosphorylation, elevated serum urea nitrogen and creatinine, and obvious histological damage of kidney such as fractured tubules in control mice, which were enhanced in HNF1ß knockdown mice. These results suggest that HNF1ß may ameliorate cisplatin nephrotoxicity in vitro and in vivo, probably through regulating NF-κB signalling pathway.

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest. CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) associated with non-alcoholic fatty liver disease (NAFLD). The effects of gestational BPA exposure on hepatic lipid accumulation in offspring are not fully understood. Here, we investigate the sex-dependent effects of gestational BPA exposure on hepatic lipid and glucose metabolism in the offspring of mice to reveal the mechanisms underlying gestational BPA exposure-associated NAFLD. Pregnant mice were administered gavage with or without 1 µg kg-1 day-1 BPA at embryonic day 7.5 (E7.5)-E16.5. Hepatic glucose and lipid metabolism were evaluated in these models. Both male and female offspring mice exhibited hepatic fatty liver after BPA treatment. Lipid accumulation and dysfunction of glucose metabolism were observed in male offspring. We revealed abnormal expression of lipid regulators in the liver and that inhibition of peroxisome proliferator-activated receptor γ (PPARγ) repressed hepatic lipid accumulation induced by gestational BPA exposure. We also found a sex-dependent decrease of hepatocyte nuclear factor 1b (HNF1b) expression in male offspring. The transcriptional repression of PPARγ by HNF1b was confirmed in L02 cells. Downregulation of HNF1b, upregulation of PPARγ, and subsequent upregulation of hepatic lipid accumulation were essential for NAFLD development in male offspring gestationally exposed to BPA as well as BPA-exposed adult male mice. Dysregulation of the HNF1b/PPARγ pathway may be involved in gestational BPA exposure-induced NAFLD in male offspring. These data provide new insights into the mechanism of gestational BPA exposure-associated sex-dependent glucose and lipid metabolic dysfunction. Graphical abstract Schematic of the mechanism of gestational BPA exposure-induced glucose and lipid metabolic dysfunction.

HNF1B inhibits cell proliferation via repression of SMAD6 expression in prostate cancer.

Prostate cancer is the most common malignancy in men in developed countries. In previous study, we identified HNF1B (Hepatocyte Nuclear Factor 1ß) as a downstream effector of Enhancer of zeste homolog 2 (EZH2). HNF1B suppresses EZH2-mediated migration of two prostate cancer cell lines via represses the EMT process by inhibiting SLUG expression. Besides, HNF1B expression inhibits cell proliferation through unknown mechanisms. Here, we demonstrated that HNF1B inhibited the proliferation rate of prostate cancer cells. Overexpression of HNF1B in prostate cancer cells led to the arrest of G1 cell cycle and decreased Cyclin D1 expression. In addition, we re-explored data from ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq), and demonstrated that HNF1B repressed Cyclin D1 via direct suppression of SMAD6 expression. We also identified CDKN2A as a HNF1B-interacting protein that would contribute to HNF1B-mediated repression of SMAD6 expression. In summary, we provide the novel mechanisms and evidence in support HNF1B as a tumour suppressor gene for prostate cancer.

HNF1B, EZH2 and ECI2 in prostate carcinoma. Molecular, immunohistochemical and clinico-pathological study.

Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target.

Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype.

BACKGROUND: We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1ß+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. METHODS: Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1ß and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. RESULTS: p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3ß, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1ß+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. CONCLUSION: These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with an HNF-1ß+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics. Video Abstract.

Pathology of clear cell carcinoma of the ovary: A basic view based on cultured cells and modern view from comprehensive approaches.

Clear cell carcinoma (CCC) is a distinct histologic type of ovarian carcinoma. CCC is more frequent in Japan than in the Western world. CCC is chemo-resistant and often associated with paraneoplastic thromboembolism. Histologically, CCC is characterized by both cancer cells and stromas, being concordant with the cytological features. Clear cells contain abundant glycogen. Hepatocyte nuclear factor-1ß is a specific marker of CCC, and is likely to be involved in glucose metabolism. Extracellular matrix (ECM)-deposited stroma and plasma cell-rich inflammatory stroma are characteristic stromas of CCC. Studies using CCC cell lines showed that CCC cells produce ECMs and stimulate plasma cell differentiation in a paracrine manner. Most CCCs, as well as endometrioid carcinomas, originate from ovarian endometriosis. This is supported by molecular genetic data, although it remains unclear why different histologic types originate from the same precursor. CCC and endometrioid carcinoma are Lynch syndrome-associated ovarian carcinomas. Recent comprehensive studies indicate that CCC is distinct not only in terms of histology but also in genomics, epigenomics and transcriptomics. This review summarizes the pathology of ovarian CCC along with a basic view based on cultured cells, and refers to recent genetic and omic data.

HNF1ß is a sensitive and specific novel marker for yolk sac tumor: a tissue microarray analysis of 601 testicular germ cell tumors.

Hepatocyte Nuclear Factor 1 beta (HNF1ß) is a transcription factor which plays an important role during early organogenesis, especially of the pancreato-biliary and urogenital tract. Furthermore, HNF1ß is an established marker in the differential diagnosis of ovarian cancer and shows a distinct nuclear expression in the clear cell carcinoma subtype. Recently, it has been described in yolk sac tumor, which represents a common component in many non-seminomatous germ cell tumors. Due to its broad histologic diversity, the diagnosis may be challenging and additional tools are very helpful in the workup of germ cell tumors. Immunohistochemistry was used to study HNF1ß expression in a tissue microarray (TMA) of 601 testicular germ cell tumors including seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, germ cell neoplasia in situ (GCNIS), and normal tissue. The expression pattern was compared to glypican 3 (GPC3) and α-fetoprotein (AFP), two markers currently in use for the detection of yolk sac tumor. HNF1ß showed a distinct nuclear staining in comparison to the cytoplasmic pattern of GPC3 and AFP. The sensitivity and specificity of HNF1ß were 85.4% and 96.5%, of GPC3 83.3% and 90.7%, of AFP 62.5% and 97.7%. We conclude that HNF1ß allows a reliable distinction of yolk sac tumor from other germ cell tumor components. Therefore, we propose HNF1ß as a novel and robust marker in the immunohistochemical workup of testicular germ cell tumors.

Hepatocyte nuclear factor-1ß suppresses the stemness and migration of colorectal cancer cells through promoting miR-200b activity.

The effects of hepatocyte nuclear factors (HNFs) have been established in various tumors; however, the roles of HNF-1ß in colorectal cancer progression are never been found. In the present study, HNF-1ß expression was initially detected in clinical tissue samples and online datasets and HNF-1ß was found to be highly expressed in colorectal cancer tissues. In addition, a positive correlation existed between HNF-1ß expression and the overall survival of patients with colorectal cancer. In vitro and in vivo experiments revealed that HNF-1ß suppressed the stemness and migration of colorectal cancer cells. Combined with microRNAs (miRNAs) based on transcriptome-sequencing analysis, mechanistic studies showed that HNF-1ß directly bound to miR-200b promoter and thus promoted miR-200b expression, this HNF-1ß/miR-200b resulted in the downregulation of the expression of miR-200b downstream effectors. Furthermore, HNF-1ß inhibits the stemness and migration of colorectal cancer cells through miR-200b. This study reveals a novel HNF-1ß/miR-200b axis responsible for the stemness of colorectal cancer cells.

Identification of novel HNF1B mRNA splicing variants and their qualitative and semi-quantitative profile in selected healthy and tumour tissues.

Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor crucial for the development of several tissues, and a promising biomarker of certain solid tumours. Thus far, two HNF1B alternative splicing variants (ASVs) have been described, however, the complete spectrum, prevalence and role of HNF1B ASVs in tumorigenesis are unclear. Considering the equivocal data about HNF1B ASVs and expression presented in literature, our aim was to characterize the spectrum of HNF1B mRNA splicing variants across different tissues. Here, we characterize HNF1B ASVs with high sensitivity in carcinomas of the uterine corpus, large intestine, kidney, pancreas, and prostate, with selected paired healthy tissues, using the previously described multiplex PCR and NGS approach. We identified 45 ASVs, of which 43 were novel. The spectrum and relative quantity of expressed ASVs mRNA differed among the analysed tissue types. Two known (3p, Δ7_8) and two novel (Δ7, Δ8) ASVs with unknown biological functions were detected in all the analysed tissues in a higher proportion. Our study reveals the wide spectrum of HNF1B ASVs in selected tissues. Characterization of the HNF1B ASVs is an important prerequisite for further expression studies to delineate the HNF1B splicing pattern, potential ASVs functional impact, and eventual refinement of HNF1B's biomarker role.

Role of transcription factor hepatocyte nuclear factor-1ß in polycystic kidney disease.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a DNA-binding transcription factor that is essential for normal kidney development. Mutations of HNF1B in humans produce cystic kidney diseases, including renal cysts and diabetes, multicystic dysplastic kidneys, glomerulocystic kidney disease, and autosomal dominant tubulointerstitial kidney disease. Expression of HNF1B is reduced in cystic kidneys from humans with ADPKD, and HNF1B has been identified as a modifier gene in PKD. Genome-wide analysis of chromatin binding has revealed that HNF-1ß directly regulates the expression of known PKD genes, such as PKHD1 and PKD2, as well as genes involved in PKD pathogenesis, including cAMP-dependent signaling, renal fibrosis, and Wnt signaling. In addition, a role of HNF-1ß in regulating the expression of noncoding RNAs (microRNAs and long noncoding RNAs) has been identified. These findings indicate that HNF-1ß regulates a transcriptional and post-transcriptional network that plays a central role in renal cystogenesis.

Hepatocyte nuclear factor 1ß suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer-binding factor 1.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1ß produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells results in activation of ß-catenin and increased expression of lymphoid enhancer-binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1ß in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1ß binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1ß decreases H3K27 trimethylation repressive marks and increases ß-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1ß recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the ß-catenin-binding domain of LEF1 in HNF-1ß-deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1ß through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1ß regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1ß.

Napsin-A and AMACR are Superior to HNF-1ß in Distinguishing Between Mesonephric Carcinomas and Clear Cell Carcinomas of the Gynecologic Tract.

Mesonephric carcinoma is a rare gynecologic neoplasm commonly mistaken for clear cell carcinoma, because of their overlapping morphologic features. Both tumors are negative for estrogen receptor and p16, magnifying this diagnostic dilemma. Recently, hepatocyte nuclear factor-1 beta (HNF-1ß), a marker for clear cell carcinoma, has also been shown to be positive in mesonephric carcinomas. Other more recent markers for clear cell carcinoma, however, such as Napsin-A and alpha-methylacyl-CoA racemase (AMACR), have not yet been studied in mesonephric carcinomas. Here we examine HNF-1ß, AMACR, and Napsin-A immunohistochemistry in 18 mesonephric and 55 endometrial/cervical clear cell carcinomas. HNF-1ß was considered positive if nuclear staining was present in ≥70% of cells and at least moderate intensity; for Napsin-A and AMACR, any cytoplasmic staining was considered positive (≥1%). H-scores were determined by multiplying the intensity score by proportion score. HNF-1ß was positive in a substantial portion of mesonephric carcinomas (9/18, 50%; H-score 98) and clear cell carcinomas (34/55, 62%; H-score 163) and did not distinguish between the 2 entities (specificity, 50%; P-value of H-score=0.08). Napsin-A and AMACR expression was significantly higher in clear cell [43/55 (78%) and 41/55 (75%), respectively] than mesonephric carcinomas [4/18 (22%) and 4/18 (22%) respectively], and helpful in this differential (specificity: 78% and 78%; P<0.05 for both). When Napsin-A and AMACR staining were seen in mesonephric carcinomas, staining was focal (≤5%), whereas staining in clear cell carcinomas was patchy/diffuse. In summary, Napsin-A and AMACR are helpful in distinguishing mesonephric carcinomas from clear cell carcinomas of the female genital tract, but HNF-1ß is not.

Use of Immunohistochemical Markers (HNF-1ß, Napsin A, ER, CTH, and ASS1) to Distinguish Endometrial Clear Cell Carcinoma From Its Morphologic Mimics Including Arias-Stella Reaction.

The diagnosis of clear cell (CC) carcinoma of the endometrium can be challenging, especially when endometrioid (EC) and serous (SC) endometrial cancers exhibit nonspecific clear cell changes, in carcinomas with mixed histology and in the setting of Arias-Stella reaction (ASR). In this study, classic CC immunohistochemical markers (Napsin A, HNF-1ß, and ER) and 2 recent novel markers, cystathionine gamma-lyase (CTH) and arginosuccinate synthase (ASS1), are assessed for their utility in distinguishing CC from its morphologic mimics. Tissue microarrays containing 64 CC, 128 EC, 5 EC with clear cell change, 16 SC, 5 mixed carcinomas, and 11 whole ASR sections were stained, with 12 additional examples of ASR stained subsequently. A cutoff of 70% and moderate intensity were used for HNF-1ß, 80% of cells and strong intensity were used for CTH, and any staining was considered positive for the remaining markers. For differentiating CC from pure EC and SC, HNF-1ß, Napsin A, and CTH all performed well. HNF-1ß had higher specificity (99.3% vs. 95.1%) but lower sensitivity (55.8% vs. 73.1%) compared with Napsin A. CTH did not substantially outperform HNF- 1ß or Napsin A (sensitivity 51.9%, specificity 99.3%). ASS1 and ER were not helpful (specificities of 60.1% and 22.6%). For differentiating CC from ASR, HNF-1ß, Napsin A, and CTH stained a large proportion of ASR and were not useful. However, ER positivity and ASS1 negativity were helpful for identifying ASR (specificity 88.2% and 95.1%, respectively). EC with clear cell changes exhibited immunohistochemical patterns similar to pure EC (HNF-1ß-, ER+, and CTH-). No markers were useful in confirming the CC components in mixed carcinomas.