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.

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.

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.

Bifunctional protein PCBD2 operates as a co-factor for hepatocyte nuclear factor 1ß and modulates gene transcription.

Hepatocyte nuclear factor 1ß (HNF1ß) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1ß-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1ß function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1ß interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1ß. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1ß the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1ß to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1ß activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1ß-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1ß-target genes involved in additional processes besides ion transport in the kidney.

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.

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.

Hepatocyte nuclear factor-1ß regulates Wnt signaling through genome-wide competition with ß-catenin/lymphoid enhancer binding factor.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a tissue-specific transcription factor that is essential for normal kidney development and renal tubular function. Mutations of HNF-1ß produce cystic kidney disease, a phenotype associated with deregulation of canonical (ß-catenin-dependent) Wnt signaling. Here, we show that ablation of HNF-1ß in mIMCD3 renal epithelial cells produces hyperresponsiveness to Wnt ligands and increases expression of Wnt target genes, including Axin2, Ccdc80, and Rnf43 Levels of ß-catenin and expression of Wnt target genes are also increased in HNF-1ß mutant mouse kidneys. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) in wild-type and mutant cells showed that ablation of HNF-1ß increases by 6-fold the number of sites on chromatin that are occupied by ß-catenin. Remarkably, 50% of the sites that are occupied by ß-catenin in HNF-1ß mutant cells colocalize with HNF-1ß-occupied sites in wild-type cells, indicating widespread reciprocal binding. We found that the Wnt target genes Ccdc80 and Rnf43 contain a composite DNA element comprising a ß-catenin/lymphoid enhancer binding factor (LEF) site overlapping with an HNF-1ß half-site. HNF-1ß and ß-catenin/LEF compete for binding to this element, and thereby HNF-1ß inhibits ß-catenin-dependent transcription. Collectively, these studies reveal a mechanism whereby a transcription factor constrains canonical Wnt signaling through direct inhibition of ß-catenin/LEF chromatin binding.

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.

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.

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.

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.

Genomic integration of ERRγ-HNF1ß regulates renal bioenergetics and prevents chronic kidney disease.

Mitochondrial dysfunction is increasingly recognized as a critical determinant of both hereditary and acquired kidney diseases. However, it remains poorly understood how mitochondrial metabolism is regulated to support normal kidney function and how its dysregulation contributes to kidney disease. Here, we show that the nuclear receptor estrogen-related receptor gamma (ERRγ) and hepatocyte nuclear factor 1 beta (HNF1ß) link renal mitochondrial and reabsorptive functions through coordinated epigenomic programs. ERRγ directly regulates mitochondrial metabolism but cooperatively controls renal reabsorption via convergent binding with HNF1ß. Deletion of ERRγ in renal epithelial cells (RECs), in which it is highly and specifically expressed, results in severe renal energetic and reabsorptive dysfunction and progressive renal failure that recapitulates phenotypes of animals and patients with HNF1ß loss-of-function gene mutations. Moreover, ERRγ expression positively correlates with renal function and is decreased in patients with chronic kidney disease (CKD). REC-ERRγ KO mice share highly overlapping renal transcriptional signatures with human patients with CKD. Together these findings reveal a role for ERRγ in directing independent and HNF1ß-integrated programs for energy production and use essential for normal renal function and the prevention of kidney disease.

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.

Computational and functional analyses of T2D GWAS SNPs for transcription factor binding.

Genome-wide association studies (GWASs) have successfully identified numerous non-coding genetic variants for type 2 diabetes (T2D), but the functional roles underlying these non-coding variants remain largely unknown. The effects of T2D GWAS lead SNPs on transcriptional factors binding motifs were firstly analyzed via JASPAR, followed by functional validations including dual-luciferase reporter assays, biotin-based DNA pull-down assays, real-time quantitative PCR, and western blotting. The results showed that GWAS SNP rs4430796 conferred T allele specific transcriptional enhancer activity via a PAX6 binding element, and upregulated the expression of HNF1B. GWAS SNP rs4607103 showed a bidirectional modulation of ADAMTS9-AS2 and ADAMTS9 by TCF7L2 in a T allele-specific manner. GWAS SNP rs849135 conferred C allele-specific bidirectional transcriptional enhancer activity via a CREB1 binding element. Our findings have uncovered the functional mechanisms of three T2D GWAS SNPs via affecting the binding of transcription factors, providing new insights into the genetics and molecular pathogenesis of T2D.

HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis.

Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.

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.

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.

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.

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.