Impact of Soy ß-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

BACKGROUND: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. AIM: To characterize the effect of two soy peptides, namely, ß-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. METHODS: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. RESULTS: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (-42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9-LDLR interaction. CONCLUSIONS: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

Intravenous nanoparticle vaccination generates stem-like TCF1+ neoantigen-specific CD8+ T cells.

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8+ T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1+PD-1+CD8+ T cells as compared to subcutaneous immunization (SNP-SC). Single-cell RNA sequencing showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade, leading to superior antitumor response as compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8+ T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8+ T cells.

Expression of liver fatty acid binding protein in hepatocellular carcinoma.

Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Prognostic value of hepatocyte nuclear factors 4α and 1α identified by tissue microarray in resectable hepatocellular carcinoma.

BACKGROUND AND AIM: This study aimed to investigate the prognostic value of expression of hepatocyte nuclear factors (HNFs) involved in hepatic gene transcription in patients undergoing curative resection for hepatocellular carcinoma (HCC). METHODS: We performed immunohistochemical analyses on microarrays of the tumors and matched adjacent tissue using antibodies against HNF1α, HNF1ß, HNF4α, and α-fetoprotein (AFP). We evaluated the prognostic value of biomarker expression using Cox regression and the Kaplan-Meier method in a training cohort of 220 patients and conducted an independent validation in 232 patients. We also determined whether measurement of HNFs improved risk prediction beyond the use of established factors, using net reclassification improvement (NRI). RESULTS: Post-surgical recurrence and hepatic death were predicted by intratumoral HNF4α underexpression in both cohorts. In the training cohort they were also predicted by peritumoral HNF1α positivity. A pooled cohort analysis showed that these predictors were independently associated with early but not late-phase recurrence, and resultant mortality. Intratumoral expression levels of HNF4α were correlated with those of HNF1α, HNF1ß, and AFP (P < 0.05). Similarly, HNF1α expression in peritumoral tissue was correlated with that of other markers (P < 0.05). There was no significant correlation between expression of HNF4α in tumors and HNF1α in peritumoral tissue. Adding combinations of intratumoral HNF4α and peritumoral HNF1α to 2-year recurrence and 5-year mortality models including known clinicopathological prognostic factors significantly improved the NRI indexes (39% and 44%, respectively; P < 0.05). CONCLUSIONS: Immunohistological activation of intratumoral HNF4α and depletion of peritumoral HNF1α have prognostic significance for delayed recurrence and death after HCC resection.

Hepatocyte nuclear factor 1ß is a novel prognostic marker independent of the Milan criteria in transplantable hepatocellular carcinoma: a retrospective analysis based on tissue microarrays.

We retrospectively investigated the prognostic value of hepatocyte nuclear factor 1 (HNF1) proteins in 159 liver transplant patients with hepatocellular carcinoma (HCC), including 36 (22.6%) exceeding the Milan criteria. The expression of alpha-fetoprotein (AFP), HNF1α, and HNF1ß was examined with immunohistochemistry on duplicate tissue microarray slides containing HCC tumor explants. The times to recurrence and cancer death were analyzed with a Cox regression model and were compared according to the expression of markers of interest. We compared risk predictions with area under the receiver operator curves (AUROCs) and C statistics. AFP, HNF1α, and HNF1ß were positive in 22.6%, 46.5%, and 61.0% of the tumor immunoprofiles, respectively. Although several variables were associated with the times to recurrence and cancer death in univariate Cox analyses, only AFP expression for the time to recurrence and the Milan criteria and HNF1ß expression for the times to recurrence and cancer death remained significant after multivariate adjustments. The expression of HNF1ß (but not HNF1α) was related to a serum AFP level ≥ 200 ng/mL, microvascular invasion, and AFP expression (P < 0.05 for all). A subgroup analysis showed that in the group meeting the Milan criteria, recurrence and cancer death rates at 10 years in the HNF1ß-negative patients were approximately one-tenth of those in the HNF1ß-positive patients, but the difference was not significant in the group exceeding the Milan criteria. The addition of HNF1ß expression to the Milan criteria increased the C statistics and AUROCs for both recurrence and mortality (P < 0.05 for all). In conclusion, the immunohistological detection of HNF1ß predicts recurrence and HCC-specific death after transplantation and provides an additive benefit in comparison with the Milan selection criteria on their own.

[Interest of immunohistochemistry for the diagnosis of benign hepatocellular tumors]. / Apport de l'immunohistochimie dans le diagnostic des tumeurs hépatocellulaires bénignes.

In this review, we focus on the interest of immunohistochemistry first, to differentiate the two types of benign hepatocellular nodules: focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) and second, to recognize the different subtypes of HCA: HNF1α inactivated HCA, ß-catenin activated HCA and inflammatory HCA. This pathomolecular classication followed the study of genotype/phenotype correlations which led to the identification of immunohistochemical data characteristic of each subgroup. Immunohistochemical characteristics described on resected specimen are suitable on biopies of these tumors, facilitating their diagnosis and therefore allowing a better management of the patients.

Hepatocyte nuclear factor 1 alpha and 4 alpha are factors involved in interindividual variability in the expression of UGT1A6 and UGT1A9 but not UGT1A1, UGT1A3 and UGT1A4 mRNA in human livers.

UDP-glucuronosyltransferases (UGTs) catalyze phase-II biotransformation reaction of a variety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual differences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1alpha and HNF4alpha mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual differences in expression levels were observed in UGT1A1 (8.6-fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1alpha and HNF4alpha mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no significant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1alpha and HNF4alpha are the factors involved in the interindividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression.

Hepatocellular adenoma subtype classification using molecular markers and immunohistochemistry.

UNLABELLED: Hepatocellular adenomas (HCA) with activated beta-catenin present a high risk of malignant transformation. To permit robust routine diagnosis to allow for HCA subtype classification, we searched new useful markers. We analyzed the expression of candidate genes by quantitative reverse transcription polymerase chain reaction QRT-PCR followed by immunohistochemistry to validate their specificity and sensitivity according to hepatocyte nuclear factor 1 alpha (HNF1alpha) and beta-catenin mutations as well as inflammatory phenotype. Quantitative RT-PCR showed that FABP1 (liver fatty acid binding protein) and UGT2B7 were downregulated in HNF1alpha-inactivated HCA (P