Albrecht Piiper

Serum miR-122 as a biomarker of necroinflammation in patients with chronic hepatitis C virus infection.

OBJECTIVES:The liver contains large amounts of microRNA-122 (miR-122), whereas other tissues contain only marginal amounts of this miRNA. MicroRNAs have also been found to circulate in the blood in a cell-free form; their potential as readily accessible disease markers is currently evaluated. Here, we investigated if the serum levels of miR-122 might be useful as disease parameter in patients with chronic hepatitis C virus (HCV) infection.METHODS:RNA was extracted from sera of patients with chronic HCV infection (CHC) and healthy controls and was analyzed for miR-22 content by quantitative real-time reverse-transcription polymerase chain reaction. miR-122 serum levels were correlated with standard parameters of liver function. Liver biopsies from the same patients were examined for the histologic activity index (HAI) and the degree of fibrosis.RESULTS:Sera from patients with CHC contained higher levels of miR-122 than sera from healthy controls. Serum miR-122 levels correlated well with markers of liver inflammatory activity, that is, the serum levels of alanine leucine transaminase (ALT) and aspartate transaminase, and the HAI score. In patients with persistently normal ALT levels, serum miR-122 levels did not differ from healthy controls. There was no correlation of serum miR-122 levels with serum albumin, international normalized ratio, liver fibrosis, or serum HCV RNA.CONCLUSIONS:The serum level of miR-122 strongly correlates with serum ALT activity and with necroinflammatory activity in patients with CHC and elevated ALT levels, but not with fibrosis stage and functional capacity of the liver.

Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma

BACKGROUND The identification of new biomarkers to predict the aggressiveness of hepatocellular carcinoma (HCC) and supplement the current set of prognosis and treatment algorithms is an important clinical need. Extracellular microRNAs (miRNAs) circulating in the blood are a new class of highly promising disease markers. AIM Here we investigated the prognostic potential of miR-1 and miR-122 in sera from patients with HCC. METHODS RNA was extracted from 195 sera of HCC patients and 54 patients with liver cirrhosis, obtained at the time of study enrolment. miR-1 and miR-122 levels were correlated with overall survival (OS), Cancer of the Liver Italian Program (CLIP) score, Barcelona Clinic Liver Cancer stage, clinical chemistry parameters and tumor specific treatment. RESULTS Patients with higher miR-1 and miR-122 serum levels showed longer OS than individuals with lower miR-1 and miR-122 serum concentrations (hazard ratio [HR] 0.440, 95% confidence interval [CI] 0.233-0.831, P=0.011 for miR-1 and HR 0.493, 95% CI 0.254-0.956, P=0.036 for miR-122, respectively). Serum miR-1 and miR-122 concentrations did not differ significantly between patients with HCC and liver cirrhosis. An age-, sex-, tumor stage and treatment-adjusted multivariate Cox regression analysis revealed that miR-1 serum levels (HR 0.451, 95% CI 0.228-0.856, P=0.015) were independently associated with OS, whereas serum miR-122 was not. miR-1 serum levels showed no relevant correlation with clinical chemistry liver parameters, whereas serum miR-122 correlated with clinical chemistry parameters of hepatic necroinflammation, liver function and synthetic capacity. CONCLUSION Our data indicate that serum miR-1 is a new independent parameter of OS in HCC patients and may therefore improve the predictive value of classical HCC staging scores.

Serum MicroRNA-21 as Marker for Necroinflammation in Hepatitis C Patients with and without Hepatocellular Carcinoma

Background MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC. Methodology/Principal Findings 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%. Conclusions/Significance The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC.

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF‐pocket and have the ability to allosterically activate phosphoinositide‐dependent protein kinase 1 (PDK1) by modulating the phosphorylation‐dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction‐displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation‐dependent conformational transitions in other proteins.

Serum microRNA-122 levels in different groups of patients with chronic hepatitis B virus infection

Summary.  miR‐122 is a liver‐specific microRNA, which also circulates in the blood. The levels of miR‐122 in serum and plasma correlate with hepatic necroinflammation in patients with hepatitis B virus (HBV) infection. Here, we investigated whether miR‐122 levels correlate with surrogate markers for viral replication and translation. Furthermore, we examined whether miR‐122 levels differ in the different groups of HBV‐infected patients and whether miR‐122 levels may be useful to identify patients with higher or lower risk for liver disease progression. Therefore, RNA was extracted from sera of therapy‐naïve patients with HBV infection (n = 89) and from healthy volunteers (n = 19). The concentration of miR‐122 was assessed by quantitative real‐time reverse‐transcription PCR. HBs antigen and HBV DNA levels were quantified as surrogate parameters for HBV replication and translation. Liver biopsies were examined according to the histological activity index and the degree of fibrosis was assessed. We found that the miR‐122 serum concentration correlated with the level of ALT, HBV DNA and HBs antigen (r = 0.259, P < 0.05; r = 0.225, P < 0.05; r = 0.508, P < 0.001, respectively). The miR‐122 serum levels discriminated the different patient groups infected with HBV from healthy subjects (P < 0.001), and inactive carrier patients with high (>3500 IU/mL) or low (<3500 IU/mL) levels of HBs antigen could be differentiated by the miR‐122 serum concentration (P < 0.05). As serum miR‐122 levels strongly correlated with HBs antigen, it might be an indicator for viral translation. Furthermore, serum miR‐122 levels discriminated HBV carrier patients with high or low risk for disease progression and may, thus, be an additional marker for risk stratification.

Tumor regression by combination antisense therapy against Plk1 and Bcl-2

Increased expression of the cell proliferation-associated polo-like kinase 1 (PLK1) and apoptosis-associated BCL-2 genes has been observed in different human malignancies. Inhibition of cell proliferation and reactivation of apoptosis are basic principles in anticancer therapy. The efficiency of this approach is often limited by insuf-ficient targeting and delivery of anticancer drugs into the tumors. Phosphorothioate antisense oligodeoxynucleotides (ODNs) directed against PLK1 and BCL-2 were administered systemically via the tail vein into nude mice bearing A549, MDA-MB-435, and Detroit562 xenografts. To enhance tumor-specific uptake and to reduce systemic toxicity of antisense ODNs membrane electroporation transfer was applied in vivo. Northern and Western blot analyses were used to assess PLK1 and BCL-2 expression. Tumor mass was assessed after resection of tumors. All three cell lines and corresponding xenografts expressed high levels of PLK1 and were sensitive towards antisense PLK1 treatment. Antisense BCL-2 therapy was effective in tumors expressing high levels of BCL-2, but not in A549 cells and corresponding xenografts, which express low levels of BCL-2. Administration of antisense ODNs in a dose of 5 mg/kg, twice weekly during four weeks supported by the membrane electroporation transfer, eradicated 60–100% of the xenografted tumors. Antitumor effect in BCL-2 overexpressing MDA-MB-435 cells was synergistic for BCL-2 and PLK1 combination therapy. This study provides evidence that combined systemic administration of antisense ODNs against proliferation and pro- survival associated targets and in vivo electroporation of tumors represents a promising antitumor therapeutic approach.

Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers

Background MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. Methods The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. Results The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Conclusions Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.

Involvement of the platelet-derived growth factor receptor in angiotensin II-induced activation of extracellular regulated kinases 1 and 2 in human mesangial cells

In mesangial cells angiotensin II (Ang II) has been shown to activate extracellular regulated kinases 1 and 2 (ERK1/2). Here, we studied the role of the epidermal growth factor receptor (EGFR) and platelet‐derived growth factor receptor (PDGFR) in Ang II‐induced ERK1/2 activation in human mesangial cells. Ang II induced activation of ERK1/2 via the AT1 receptor, and this response was blocked by the PDGFR‐selective tyrosine kinase inhibitor AG1295, but not by AG1478, an EGFR‐selective tyrosine kinase inhibitor, indicating participation of the PDGFR, but not of the EGFR in Ang II‐induced ERK1/2 activation. In agreement with this assumption, Ang II caused tyrosine phosphorylation of the PDGFR and the adapter protein Shc in an AG1295‐sensitive fashion. In conclusion, our data show that Ang II‐induced activation of mitogenic signalling cascade in human mesangial cells involves ligand‐independent activation of the PDGFR, but not of the coexpressed EGFR.

Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells

Epithelial‐mesenchymal transition (EMT) is an important mechanism to initiate cancer invasion and metastasis. Bone morphogenetic protein (BMP)‐9 is a member of the transforming growth factor (TGF)‐β superfamily. It has been suggested to play a role in cancer development in some non‐hepatic tumors. In the present study, two hepatocellular carcinoma (HCC) lines, HLE and HepG2, were treated with BMP‐9 in vitro, and phenotypic changes and cell motility were analyzed. In situ hybridization (ISH) and immunohistochemical analyses were performed with human HCC tissue samples in order to assess expression levels of BMP‐9. In vivo, BMP‐9 protein and mRNA were expressed in all the tested patients to diverse degrees. At the protein level, mildly positive (1 + ) BMP‐9 staining could be observed in 25/41 (61%), and moderately to strongly positive (2 + ) in 16/41 (39%) of the patients. In 27/41 (65%) patients, the BMP‐9 protein expression level was consistent with the mRNA expression level as measured by ISH. In those patients with 2 + protein level, nuclear pSmad1 expression in cancer cells was also significantly increased. Expression of BMP‐9 was positively related to nuclear Snail expression and reversely correlated to cell surface E‐cadherin expression, although this did not reach statistical significance. Expression levels of BMP‐9 were significantly associated with the T stages of the investigated tumors and high levels of BMP‐9 were detected by immunofluorescence especially at the tumor borders in samples from an HCC mouse model. In vitro, BMP‐9 treatment caused a reduction of E‐cadherin and ZO‐1 and an induction of Vimentin and Snail expression. Furthermore, cell migration was enhanced by BMP‐9 in both HCC cell lines. These results imply that EMT induced by BMP‐9 is related to invasiveness of HCC.

Serum MicroRNA-122 Predicts Survival in Patients with Liver Cirrhosis

Background Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease. Aim Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis. Methods 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis. Results Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age. Conclusions Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis.