Metformin and exenatide upregulate hepatocyte nuclear factor-4α, sex hormone binding globulin levels and improve hepatic triglyceride deposition in polycystic ovary syndrome with insulin resistance rats.

OBJECTIVE: To explore the efficacy and underlying mechanisms of metformin and exenatide in reversing reproductive and metabolic disturbances in letrozole combined with high-fat diet-induced polycystic ovary syndrome (PCOS) rats. METHODS: Rats with PCOS and insulin resistance (IR) were induced by intra-gastric instillation of letrozole combined with a high-fat diet and verified by histological screening of vaginal exfoliated cells. After metformin and exenatide supplementation, body weight, chow intake and ovarian morphology were observed. Serum biochemical profiles were analyzed using ELISA, while the levels of key anabolism-related proteins, including sex hormone binding globulin (SHBG), hepatocyte nuclear factor-4α (HNF-4α), PI3K, and AKT, were determined using western blotting. RESULTS: The estrus cycle and ovarian morphology of rats with PCOS and IR were significantly recovered following metformin and exenatide treatment, with decreased body weight and chow intake. Furthermore, PCOS-induced changes in metabolic disorders including IR and hepatic triglyceride (TG) deposition, and hyperandrogenemia were reversed by treatment with both drugs. Specifically, the levels of HNF-4α and SHBG in liver tissue of rats with PCOS and IR were upregulated significantly. CONCLUSIONS: Both metformin and exenatide could recover the estrous cycle and ovarian morphology, reduce body weight and high-fat chow intake, and improve glycolipid metabolism disorders and hyperandrogenemia in PCOS with IR rat models. Interestingly, our findings also highlight the potential of both therapeutic agents for improving IR by regulating the liver PI3K/AKT pathway, reducing the deposition of hepatic TG, as well as upregulating the levels of SHBG and HNF-4α in PCOS with IR rat liver tissue.

Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation.

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to limited therapeutic options. This study examines the roles of genome-wide association study identified PDAC-associated genes as therapeutic targets. We have identified HNF4G gene whose silencing most effectively repressed PDAC cell invasiveness. HNF4G overexpression is induced by the deficiency of transcriptional factor and tumor suppressor SMAD4. Increased HNF4G are correlated with SMAD4 deficiency in PDAC tumor samples and associated with metastasis and poor survival time in xenograft animal model and in patients with PDAC (log-rank P = 0.036; HR = 1.60, 95% CI = 1.03-2.47). We have found that Metformin suppresses HNF4G activity via AMPK-mediated phosphorylation-coupled ubiquitination degradation and inhibits in vitro invasion and in vivo metastasis of PDAC cells with SMAD4 deficiency. Furthermore, Metformin treatment significantly improve clinical outcomes and survival in patients with SMAD4-deficient PDAC (log-rank P = 0.022; HR = 0.31, 95% CI = 0.14-0.68) but not in patients with SMAD4-normal PDAC. Pathway analysis shows that HNF4G may act in PDAC through the cell-cell junction pathway. These results indicate that SMAD4 deficiency-induced overexpression of HNF4G plays a critical oncogenic role in PDAC progression and metastasis but may form a druggable target for Metformin treatment.

LncRNA LINP1 regulates acute myeloid leukemia progression via HNF4α/AMPK/WNT5A signaling pathway.

LncRNAs play critical roles in various pathophysiological and biological processes, such as protein translation, RNA splicing, and epigenetic modification. Indeed, abundant evidences demonstrated that lncRNA act as competing endogenous RNAs (ceRNAs) to participate in tumorigenesis. However, little is known about the underlying function of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) in pediatric and adolescent acute myeloid leukemia (AML). The expression of LINP1 was examined in AML patient samples by qRT-PCR. Cell proliferation was examined by CCK-8 and Edu assays. ß-Galactosidase senescence assay, mGlucose uptake assay, lactate production assay, and Gene Ontology (GO) analysis were performed for functional analysis. We found that LINP1 was significantly overexpressed in AML patients at diagnosis, whereas downregulated after complete remission (CR). Furthermore, knockdown of LINP1 expression remarkably suppressed glucose uptake and AML cell maintenance. Mechanistically, LINP1 was found to inhibit the glucose metabolism by suppressing the expression of HNF4a. Both LINP1 and HNF4a knockdown reduced the expression levels of AMPK phosphorylation and WNT5A, indicating for the first time that LINP1 strengthened the HNF4a-AMPK/WNT5A signaling pathway involved in cell glucose metabolism modulation and AML cell survival. Taken together, our results indicated that LINP1 promotes the malignant phenotype of AML cells and stimulates glucose metabolism, which can be regarded as a potential prognostic marker and therapeutic target for AML.

Targeting PRMT5 Activity Inhibits the Malignancy of Hepatocellular Carcinoma by Promoting the Transcription of HNF4α.

Background: Liver cancer stem cells (LCSCs) are responsible for the initiation, progression and chemoresistance of liver cancer. However, no agent targeting LCSC is available in the clinic to date. Here, we investigated the effects of targeting protein arginine methyltransferase 5 (PRMT5), an epigenetic regulator, on LCSCs and HCC using a novel PRMT5 inhibitor DW14800. Methods: Tumor spheroid formation culture was used to enrich LCSCs and assess their self-renewal capability. Human alpha-1-antitrypsin (A1AT) ELISA, acetylated low-density lipoprotein (ac-LDL) uptake, periodic acid-Schiff (PAS) reactions and senescence associated ß-galactosidase (SA-ß-gal) activity assays were performed to examine the differentiation status of HCC cells. The effects of DW14800 on HCC malignancy were assessed in HCC cell lines and on an HCC xenograft model in mice. Chromatin immunoprecipitation was applied to clarify the transcriptional regulation of HNF4α by PRMT5-mediated Histone H4 arginine-3 symmetrical dimethylation (H4R3me2s). Results: Quantitative real-time PCR revealed that the expression of PRMT5 was upregulated in LCSCs. DW14800 specifically decreased the symmetrical dimethylation of arginine residues in HCC cells. Treatment of DW14800 suppressed the self-renewal capacity of LCSCs while re-establishing hepatocyte-specific characteristics in HCC cells. DW14800 displayed antitumor effects in HCC cells in vitro and in xenograft HCC in vivo. Importantly, ChIP assay showed that PRMT5 and H4R3me2s bound to the promoter region of HNF4α gene, and DW14800 increased the expression of HNF4α via reducing the H4R3me2s levels and enhancing the transcription of HNF4α. Conclusions: Our data revealed the significance of targeting PRMT5 activity in LCSC elimination and HCC differentiation, and proposed that DW14800 may represent a promising therapeutic agent for HCC in the clinic.

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers.

Hepatocyte nuclear factor 4α (HNF4α) is a dimeric transcription factor that controls as much as 60% of all liver genes. However, how it achieves such broad functional diversity is unknown. Here, we show that inflammation and immune pathway genes are differentially regulated in an isoform-dependent manner, confirming that each isoform homodimer preferentially regulates a subset of HNF4α targets. With all 12 human HNF4α isoform clones, we tested combinatorial pairings to determine whether isoform heterodimers are functional. Indeed, synergistic and potent pairing combinations of isoform heterodimers were noted for HNF4α3-8, HNF4α6-12, and HNF4α5-8 that activated CYP7A1, IL6, and IL17A genes, respectively. Surprisingly, these genes are not at all activated by their corresponding isoform homodimers, suggesting that a particular heterodimer pair can regulate its own subset of target genes. Given the combinatorial possibility of 66 isoform heterodimers, our data provide the basis for a more detailed understanding of the diverse influence of HNF4α.

MiR-15/16 mediate crosstalk between the MAPK and Wnt/ß-catenin pathways during hepatocyte differentiation from amniotic epithelial cells.

MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/ß-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the ß-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate ß-catenin, and the activated ß-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.

GATA6-positive lung adenocarcinomas are associated with invasive mucinous adenocarcinoma morphology, hepatocyte nuclear factor 4α expression, and KRAS mutations.

AIMS: GATA6 is known to play a role in lung development. However, its role in the carcinogenesis of lung cancer is not well studied. The aim of this study was to analyse GATA6 expression in lung adenocarcinomas (LAs) by immunohistochemistry (IHC) in order to define its association with clinicopathological characteristics. METHODS AND RESULTS: IHC analysis of GATA6 was performed with tissue microarray slides containing 348 LAs. The association between GATA6 expression and clinicopathological parameters was evaluated. GATA6 expression in epithelial tumours other than lung cancer was also evaluated. GATA6 expression was found in 47 LAs (13.5%). This occurred more frequently in younger patients (P = 0.005), and was associated with the absence of lymph node metastasis (P =0.024), well-differentiated to moderately differentiated tumours (P < 0.001), the absence of lymphatic invasion (P = 0.020), and the absence of vascular invasion (P = 0.011). GATA6 expression was associated with mucin production (P < 0.001), the invasive mucinous adenocarcinoma subtype (P < 0.001), KRAS mutations (P = 0.026), expression of MUC2 (P < 0.001), CDX2 (P = 0.049), and MUC5AC (P < 0.001), and absence of expression of TTF-1 (P = 0.002). GATA6 expression was also associated with hepatocyte nuclear factor 4α (HNF4α) expression (P < 0.001). GATA6 expression tended to indicate better prognoses, whereas patients with HNF4α expression had significantly worse prognoses (P = 0.033). Of 270 tumours other than lung cancer, 110 expressed GATA6. CONCLUSIONS: These findings suggest that GATA6 might interact with HNF4α and contribute to the development of mucinous-type LAs.

Role of conventional immunomarkers, HNF4-α and SATB2, in the differential diagnosis of pulmonary and colorectal adenocarcinomas.

AIMS: Pulmonary (ADC) and colorectal (CRC) adenocarcinomas are frequent entities in pathological routine diagnostics. Whereas the differential diagnosis is usually straightforward based on histomorphology, it can be challenging in small biopsies. In general, CDX-2, CK20, Napsin-A and TTF-1 are recommended immunohistological markers in this scenario. Hepatocyte nuclear factor 4 alpha (HNF4-α) and special AT-rich sequence-binding protein 2 (SATB2) were described recently as promising additional markers, but comprehensive large-scale data are lacking so far. Therefore, we analysed the expression of these six markers in 1021 non-small-cell lung cancers (NSCLC), including 472 ADC as well as in 80 pulmonary metastases of CRC. METHODS AND RESULTS: Tissue microarrays of NSCLC and pulmonary metastases of CRC were stained for CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 and staining results were correlated with clinicopathological variables. ADC exhibited expression of CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 in nine (2%), 21 (4%), 17 (4%), 345 (73%), 35 (7%) and 408 (86%) samples, while 80 CRC were positive in 79 (99%), 74 (93%), 77 (96%), no (0%), 78 (98%) and five (6%) cases, respectively. CONCLUSIONS: In addition to conventional immunomarkers, HNF4-α and particularly SATB2 may be helpful in the differential diagnosis of pulmonary ADC and metastases of CRC.

Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.

Novel insights into systemic autoimmune rheumatic diseases using shared molecular signatures and an integrative analysis.

We undertook this study to identify DNA methylation signatures of three systemic autoimmune rheumatic diseases (SARDs), namely rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis, compared to healthy controls. Using a careful design to minimize confounding, we restricted our study to subjects with incident disease and performed our analyses on purified CD4+ T cells, key effector cells in SARD. We identified differentially methylated (using the Illumina Infinium HumanMethylation450 BeadChip array) and expressed (using the Illumina TruSeq stranded RNA-seq protocol) sites between cases and controls, and investigated the biological significance of this SARD signature using gene annotation databases. We recruited 13 seropositive rheumatoid arthritis, 19 systemic sclerosis, 12 systemic lupus erythematosus subjects, and 8 healthy controls. We identified 33 genes that were both differentially methylated and expressed (26 over- and 7 under-expressed) in SARD cases versus controls. The most highly overexpressed gene was CD1C (log fold change in expression = 1.85, adjusted P value = 0.009). In functional analysis (Ingenuity Pathway Analysis), the top network identified was lipid metabolism, molecular transport, small molecule biochemistry. The top canonical pathways included the mitochondrial L-carnitine shuttle pathway (P = 5E-03) and PTEN signaling (P = 8E-03). The top upstream regulator was HNF4A (P = 3E-05). This novel SARD signature contributes to ongoing work to further our understanding of the molecular mechanisms underlying SARD and provides novel targets of interest.

Hypoxia reduces HNF4α/MODY1 protein expression in pancreatic ß-cells by activating AMP-activated protein kinase.

Hypoxia plays a role in the deterioration of ß-cell function. Hepatocyte nuclear factor 4α (HNF4α) has an important role in pancreatic ß-cells, and mutations of the human HNF4A gene cause a type of maturity-onset diabetes of the young (MODY1). However, it remains unclear whether hypoxia affects the expression of HNF4α in ß-cells. Here, we report that hypoxia reduces HNF4α protein expression in ß-cells. Hypoxia-inducible factor was not involved in the down-regulation of HNF4α under hypoxic conditions. The down-regulation of HNF4α was dependent on the activation of AMP-activated protein kinase (AMPK), and the reduction of HNF4α protein expression by metformin, an AMPK activator, and hypoxia was inhibited by the overexpression of a kinase-dead (KD) form of AMPKα2. In addition, hypoxia decreased the stability of the HNF4α protein, and the down-regulation of HNF4α was sensitive to proteasome inhibitors. Adenovirus-mediated overexpression of KD-AMPKα2 improved insulin secretion in metformin-treated islets, hypoxic islets, and ob/ob mouse islets. These results suggest that down-regulation of HNF4α could be of importance in ß-cell dysfunction by hypoxia.

The role of hepatocyte nuclear factor 4-alpha in perfluorooctanoic acid- and perfluorooctanesulfonic acid-induced hepatocellular dysfunction.

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.

Hepatocyte nuclear factor 1 coordinates multiple processes in a model of intestinal epithelial cell function.

Mutations in hepatocyte nuclear factor 1 transcription factors (HNF1α/ß) are associated with diabetes. These factors are well studied in the liver, pancreas and kidney, where they direct tissue-specific gene regulation. However, they also have an important role in the biology of many other tissues, including the intestine. We investigated the transcriptional network governed by HNF1 in an intestinal epithelial cell line (Caco2). We used chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays and confirmed by siRNA-mediated depletion of HNF1, followed by RT-qPCR. Gene ontology process enrichment analysis of the HNF1 targets identified multiple processes with a role in intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites were also occupied by other members of the intestinal transcriptional network, including hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 in Caco2 cells increases FOXA2 abundance and decreases levels of CDX2, illustrating the coordinated activities of the network. These data suggest that HNF1 plays an important role in regulating intestinal epithelial cell function, both directly and through interactions with other intestinal transcription factors.

Resetting the transcription factor network reverses terminal chronic hepatic failure.

The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement.

In vitro reprogramming of pancreatic alpha cells towards a beta cell phenotype following ectopic HNF4α expression.

There is currently a shortage of organ donors available for pancreatic beta cell transplantation into diabetic patients. An alternative source of beta cells is pre-existing pancreatic cells. While we know that beta cells can arise directly from alpha cells during pancreatic regeneration we do not understand the molecular basis for the switch in phenotype. The aim of the present study was to investigate if hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor essential for a normal beta cell phenotype, could induce the reprogramming of alpha cells towards potential beta cells. We utilised an in vitro model of pancreatic alpha cells, the murine αTC1-9 cell line. We initially characterised the αTC1-9 cell line before and following adenovirus-mediated ectopic expression of HNF4α. We analysed the phenotype at transcript and protein level and assessed its glucose-responsiveness. Ectopic HNF4α expression in the αTC1-9 cell line induced a change in morphology (1.7-fold increase in size), suppressed glucagon expression, induced key beta cell-specific markers (insulin, C-peptide, glucokinase, GLUT2 and Pax4) and pancreatic polypeptide (PP) and enabled the cells to secrete insulin in a glucose-regulated manner. In conclusion, HNF4α reprograms alpha cells to beta-like cells.

Decreased expression of hepatocyte nuclear factor 4α (Hnf4α)/microRNA-122 (miR-122) axis in hepatitis B virus-associated hepatocellular carcinoma enhances potential oncogenic GALNT10 protein activity.

MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed that GALNT10 indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis.

Prevention of diabetes-promoted colorectal cancer by (n-3) polyunsaturated fatty acids and (n-3) PUFA mimetic.

The global obesity / diabetes epidemic has resulted in robust increase in the incidence of colorectal cancer (CRC). Epidemiological, animal and human studies have indicated efficacy of (n-3) PUFA in chemoprevention of sporadic and genetic-driven CRC. However, diabetes-promoted CRC presents a treatment challenge that surpasses that of sporadic CRC. This report analyzes the efficacy of (n-3) PUFA generated by the fat-1 transgene that encodes an (n-6) to (n-3) PUFA desaturase, and of synthetic (n-3) PUFA mimetic (MEDICA analog), to suppress CRC development in carcinogen-induced diabetes-promoted animal model. Carcinogen-induced CRC is shown here to be promoted by the diabetes context, in terms of increased aberrant crypt foci (ACF) load, cell proliferation and epithelial dedifferentiation, being accompanied by increase in the expression of HNF4α, ß-catenin, and ß-catenin-responsive genes. Incorporating the fat-1 transgene in the diabetes context, or oral MEDICA treatment, resulted in ameliorating the diabetic phenotype and in abrogating CRC, with decrease in ACF load, cell proliferation and the expression of HNF-4α, ß-catenin, and ß-catenin-responsive genes. The specificity of (n-3) PUFA in abrogating CRC development, as contrasted with enhancing CRC by (n-6) PUFA, was similarly verified in CRC cell lines. These findings may indicate prospective therapeutic potential of (n-3) PUFA or MEDICA in the management of CRC, in particular diabetes-promoted CRC.

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

Hepatocyte nuclear factor 4α (HNF4α) in coordination with retinoic acid receptors increases all-trans-retinoic acid-dependent CYP26A1 gene expression in HepG2 human hepatocytes.

CYP26A1 expression is very highly induced by retinoic acid (RA) in the liver, compared to most other tissues, suggesting that a liver-enriched factor may be required for its physiological transcriptional response. HNF4α is a highly conserved liver-specific/enriched member of nuclear receptor superfamily. In this study, we hypothesized that HNF4α and RARs may cooperate in an RA-dependent manner to induce a high level of CYP26A1 expression in liver cells. Partial inhibition of endogenous HNF4α by siRNA reduced the level of RA-induced CYP26A1 mRNA in HepG2 cells. Cotransfection of HNF4α, with or without RARs, demonstrated RA-dependent activation of a human CYP26A1 promoter-luciferase construct. Analysis of a 2.5-kbp putative CYP26A1 promoter sequence identified five potential HNF4α DNA response elements: H1 located in a proximal region overlapping with an RAR element-1 (RARE1 or R1); H2 and H3 in the distal region, close to RARE2 (R2) and RARE3 (R3); and H4 and H5 in intermediary regions. In EMSA and ChIP analyses HNF4α and RARs binding in the proximal and distal CYP26A1 promoter regions was significantly higher in RA-treated cells. Mutational analysis of the individual HNF4α DNA-response elements identified H1 as the major site for HNF4α binding because mutation of H1 inhibited the promoter activity by ~90%, followed by H2 mutation with less than 40% inhibition. Our results indicate that HNF4α coordinates with RARs in an RA-dependent manner to strongly induce CYP26A1 gene expression in the liver, which may explain the high level of response to RA observed in vivo.