Laura Beretta

Circulating MicroRNAs in Patients with Chronic Hepatitis C and Non-Alcoholic Fatty Liver Disease

MicroRNAs miR-122, miR-34a, miR-16 and miR-21 are commonly deregulated in liver fibrosis and hepatocellular carcinoma. This study examined whether circulating levels of these miRNAs correlate with hepatic histological disease severity in patients with chronic hepatitis C infection (CHC) or non-alcoholic fatty-liver disease (NAFLD) and can potentially serve as circulating markers for disease stage assessment. We first used an in vitro model of hepatitis C virus (HCV) infection to measure the extracellular levels of these four miRNAs. Whereas miR-21 extracellular levels were unchanged, extracellular levels of miR-122, miR-34a and to a lesser extent miR-16, steadily increased during the course of HCV infection, independently of viral replication and production. Similarly, in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher than in control individuals, while miR-21 levels were unchanged. There was no correlation between the serum levels of any of these microRNAs and HCV viral loads. In contrast, miR-122 and miR-34a levels positively correlated with disease severity. Identical results were obtained in an independent cohort of CHC patients. We extended the study to patients with NAFLD. As observed in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher in NAFLD patients than in controls, while miR-21 levels were unchanged. Again, miR-122 and miR-34a levels positively correlated with disease severity from simple steatosis to steatohepatitis. In both CHC and NAFLD patient groups, serum levels of miR-122 and miR-34a correlated with liver enzymes levels, fibrosis stage and inflammation activity. miR-122 levels also correlated with serum lipids in NAFLD patients. Conclusion: Serum levels of miR-34a and miR-122 may represent novel, noninvasive biomarkers of diagnosis and histological disease severity in patients with CHC or NAFLD.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

Identification of Osteopontin as a Novel Marker for Early Hepatocellular Carcinoma

The aim of this study was to identify a biomarker that could improve alpha‐fetoprotein (AFP) performance in hepatocellular carcinoma (HCC) surveillance among patients with cirrhosis. We performed proteomic profiling of plasma from patients with cirrhosis or HCC and validated selected candidate HCC biomarkers in two geographically distinct cohorts to include HCC of different etiologies. Mass spectrometry profiling of highly fractionated plasma from 18 cirrhosis and 17 HCC patients identified osteopontin (OPN) as significantly up‐regulated in HCC cases, compared to cirrhosis controls. OPN levels were subsequently measured in 312 plasma samples collected from 131 HCC patients, 76 cirrhosis patients, 52 chronic hepatitis C (CHC) and B (CHB) patients, and 53 healthy controls in two independent cohorts. OPN plasma levels were significantly elevated in HCC patients, compared to cirrhosis, CHC, CHB, or healthy controls, in both cohorts. OPN alone or in combination with AFP had significantly better area under the receiver operating characteristic curve, compared to AFP, in comparing cirrhosis and HCC in both cohorts. OPN overall performance remained higher than AFP in comparing cirrhosis and the following HCC groups: HCV‐related HCC, HBV‐associated HCC, and early HCC. OPN also had a good sensitivity in AFP‐negative HCC. In a pilot prospective study including 22 patients who developed HCC during follow‐up, OPN was already elevated 1 year before diagnosis. Conclusion: OPN was more sensitive than AFP for the diagnosis of HCC in all studied HCC groups. In addition, OPN performance remained intact in samples collected 1 year before diagnosis. (HEPATOLOGY 2012)

Association of Mortalin (HSPA9) with Liver Cancer Metastasis and Prediction for Early Tumor Recurrence

Hepatocellular carcinoma (HCC) is well known for poor prognosis and short survival because of high recurrence rate even after curative surgery. Today there is no available biomarker or biochemical test to indicate HCC recurrence, and this study aims to identify protein markers that can discriminate postoperative patients with early recurrence (ER), i.e. disease relapsed within the first year. In this study, 103 hepatitis B-related HCC patients were recruited, and 68 of them were used for ER-related biomarker discovery study. Proteomic expression patterns of matched tumor and adjacent non-tumor tissues from these patients plus 16 normal liver tissues were delineated by the two-dimensional gel electrophoresis differential profiling method. Significant protein spots were evaluated by hierarchical clustering analysis. SSP4612 that yielded the highest receiver operating characteristic (ROC) curve value for the ER subgroup of HCC was subsequently identified by tandem mass spectrometry, and the corresponding expression patterns were further confirmed by quantitative PCR, Western blot, and immunohistochemistry. Correlation analysis with clinicopathological data was also examined. Proteomic profiling analysis revealed overexpression of mortalin (gene HSPA9) in HCC when compared with the non-tumor and normal liver tissues (area under the curve (AUC) = 0.821). Furthermore, elevated mortalin level was also detected in the ER subgroup of HCC versus the recurrence-free state (where no cancer recurs for >1 year) (AUC = 0.833, sensitivity = 90.9%, specificity = 71.4%). Metastatic HCC cell lines also exhibited higher levels of mortalin and HSPA9 mRNA. Clinically, mortalin overexpression in HCC was closely associated with advanced tumor stages and venous infiltration, having implications for increased malignancy and aggressive behavior. Mortalin (HSPA9) is associated with HCC metastasis and thus suggested as a tumor marker for predicting early recurrence, which may have immediate clinical applications for cancer surveillance after curative surgery.

Proteomic and lipidomic signatures of lipid metabolism in NASH-associated hepatocellular carcinoma

Nonalcoholic steatohepatitis (NASH) is a common preneoplastic condition of hepatocellular carcinoma (HCC). Mice with hepatocytic deletion of Pten develop NASH and HCC later in life. This model is highly valuable for studies aimed at identifying the molecular mechanism by which metabolic disorders contribute to tumor development. We applied proteomic and lipidomic profiling approaches to Pten-null NASH liver and tumors. Circulating fatty acid composition was also characterized in these mice. The relevance to human NASH and HCC was further validated. This integrative proteomic and lipidomic study from mouse to human and from liver to blood identified the following disease signatures: (i) an HCC signature: upregulated hepatic scd1/scd2, fads2, and acsl5:acsl1 ratio, elevated vaccenic and erucic acids, and reduced margaric and linoleic acids in both liver and plasma; (ii) a NASH signature that correlates with tumor burden: upregulated hepatic elovl6, elevated oleic, adrenic, and osbond acids, and reduced cervonic acid in liver and plasma; and (iii) a NASH signature: reduced hepatic and circulating lignoceric and eicosapentaenoic acids. Altogether, these results show the role of lipid-modifying enzymes converting saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA) in HCC and the importance of an increased ratio of long chain n6-polyunsaturated fatty acids over n3-polyunsaturated fatty acids in NASH and HCC risk. They also highlight the relevance of the Pten-null model for studies related to NASH and HCC and show that circulating lipid metabolome provides a direct read of lipid changes in the liver. Most importantly, novel candidate targets for HCC diagnosis, therapy, risk assessment, and prevention were identified.

The heat shock cognate protein 70 is associated with hepatitis C virus particles and modulates virus infectivity.

There is growing evidence that virus particles contain host cell proteins. These proteins may provide viruses with means to evade the immune system or with mechanisms for cell entry and release. A proteomic analysis performed on highly purified hepatitis C virus (HCV) J6/JFH virions identified the heat shock cognate protein 70 (HSC70) as part of the viral particles. These results were further validated via immunogold electron microscopy. The HSC70 interaction HPD motif was found present on the E2 envelope of the J6/JFH strain, as well as in over 50% of genotype 2 clinical HCV isolates. In addition, HSC70 was found associated with viral particles from an HCV genotype 2a–infected patient. Preincubation of HCV particles with anti‐HSC70 antibodies decreased viral infectivity. Within infected cells, colocalization of HSC70 with the HCV core and E2 proteins was observed around lipid droplets. Reduction of HSC70 expression using an RNA interference approach decreased the volume of lipid droplets as well as viral release without affecting HCV replication levels. Conclusion: These results suggest that HSC70 modulates HCV infectivity and lipid droplet–dependent virus release. (HEPATOLOGY 2009.)

Proteomic expression signature distinguishes cancerous and nonmalignant tissues in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive liver cancer but clinically validated biomarkers that can predict natural history of malignant progression are lacking. The present study explored the proteome-wide patterns of HCC to identify biomarker signature that could distinguish cancerous and nonmalignant liver tissues. A retrospective cohort of 80 HBV-associated HCC was included and both the tumor and adjacent nontumor tissues were subjected to proteome-wide expression profiling by 2-DE method. The subjects were randomly divided into the training (n = 55) and validation (n = 25) subsets, and the data analyzed by classification-and-regression tree algorithm. Protein markers were characterized by MALDI-ToF/MS and confirmed by immunohistochemistry, Western blotting and qPCR assays. Proteomic expression signature composed of six biomarkers (haptoglobin, cytochrome b5, progesterone receptor membrane component 1, heat shock 27 kDa protein 1, lysosomal proteinase cathepsin B, keratin I) was developed as a classifier model for predicting HCC. We further evaluated the model using both leave-one-out procedure and independent validation, and the overall sensitivity and specificity for HCC both are 92.5%, respectively. Clinical correlation analysis revealed that these biomarkers were significantly associated with serum AFP, total protein levels and the Ishaks score. The described model using biomarker signatures could accurately distinguish HCC from nonmalignant tissues, which may also provide hints on how normal hepatocytes are transformed to malignant state during tumor progression.

miR-21 inhibition reduces liver fibrosis and prevents tumor development by inducing apoptosis of CD24+ progenitor cells

miR-21 is upregulated in hepatocellular carcinoma and intrahepatic cholangiocarcinoma, where it is associated with poor prognosis. Here, we offer preclinical evidence that miR-21 offers a therapeutic and chemopreventive target in these liver cancers. In mice with hepatic deletion of Pten, anti-miR-21 treatment reduced liver tumor growth and prevented tumor development. These effects were accompanied with a decrease in liver fibrosis and a concomitant reduction of CD24(+) liver progenitor cells and S100A4(+) cancer-associated stromal cells. Notch2 inhibition also occurred in tumors following anti-miR-21 treatment. We further showed that miR-21 is necessary for the survival of CD24(+) progenitor cells, a cellular phenotype mediated by Notch2, osteopontin, and integrin αv. Our results identify miR-21 as a key regulator of tumor-initiating cell survival, malignant development, and growth in liver cancer, highlighting the role of CD24(+) cells in the expansion of S100A4(+) cancer-associated stromal cells and associated liver fibrosis.

Comprehensive and quantitative proteome profiling of the mouse liver and plasma

We report a comprehensive and quantitative analysis of the mouse liver and plasma proteomes. The method used is based on extensive fractionation of intact proteins, further separation of proteins based on their abundance and size, and high‐accuracy mass spectrometry. This analysis reached a depth in proteomic profiling not reported to date for a mammalian tissue or a biological fluid, with 7099 and 4727 proteins identified with high confidence in the liver and in the corresponding plasma, respectively. This method allowed for the identification in both compartments of low‐abundance proteins such as cytokines, chemokines, and receptors and for the detection in plasma of proteins in the pg/mL concentration range. This method also allowed for semiquantitation of all identified proteins. The calculated abundance scores correlated with the abundance of the corresponding transcripts for the large majority of the proteins identified in the liver. Finally, comparison of the liver and plasma datasets demonstrated that a significant number of proteins identified in the liver can be detected in plasma. These included proteins involved in complement and coagulation, in fatty acid, purine and pyruvate metabolism, in gluconeogenesis and glycolysis, in protein ubiquitination, and in insulin, interleukin‐4, epidermal growth factor, and platelet‐derived growth factor signaling. Conclusion: This in‐depth analysis of the mouse liver and corresponding plasma proteomes provides a strong basis for investigations of liver pathobiology and biology that employ mouse models of hepatic diseases in an effort to better understand, diagnose, treat, and prevent human hepatic diseases. (HEPATOLOGY 2008.)

Mammalian target of rapamycin activation impairs hepatocytic differentiation and targets genes moderating lipid homeostasis and hepatocellular growth

The mammalian target of rapamycin (mTOR) pathway, a major regulator of translation, is frequently activated in hepatocellular carcinomas. We investigated the effects of mTOR activation in the human HepaRG cells, which possess potent hepatocytic differentiation capability. Differentiation of HepaRG cells into functional and polarized hepatocyte-like cells correlated with a decrease in mTOR and Akt activities. Stable cell lines expressing an activated mutant of mTOR were generated. Sustained activation of mTOR impaired the hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, and reduced secretion of alpha1-antitrypsin. An inhibitor of mTOR, rapamycin, was able to revert this phenotype. Furthermore, increased mTOR activity in HepaRG cells resulted in their resistance to the antiproliferative effects of transforming growth factor-beta1. Profiling of polysome-bound transcripts indicated that activated mTOR specifically targeted genes posttranscriptionally regulated on hepatocytic differentiation. Three major biological networks targeted by activated mTOR were identified: (a) cell death associated with tumor necrosis factor superfamily members, IFNs and caspases; (b) lipid homeostasis associated with the transcription factors PPARalpha, PPARdelta, and retinoid X receptor beta; and (c) liver development associated with CCAAT/enhancer binding protein alpha and hepatic mitogens. In conclusion, increased mTOR activity conferred a preneoplastic phenotype to the HepaRG cells by altering the translation of genes vital for establishing normal hepatic energy homeostasis and moderating hepatocellular growth.