Protein In Hepatocellular Carcinoma Research Articles

Polyclonal antibody preparation against candidate tumour suppressor protein MIP for detection of its expression and localization in hepatocellular carcinoma

ABSTRACTMIP is a candidate tumour suppressor protein found in hepatocellular carcinoma. It can interact with different proteins in different subcellular locations. However, antibodies against MIP for testing purposes are still not available and this has hindered the research on MIP protein. In this study, the full coding sequence of MIP protein was cloned into the prokaryotic expression vector pET-28a. The recombinant protein his-MIP was expressed by IPTG (isopropyl β-D-1-thiogalactopyranoside) induction and purified by nickel-affinity chromatography, then used to immunize New Zealand rabbits to obtain a polyclonal antibody against MIP. The sensitivity and specificity of the antibody were evaluated by enzyme-linked immunosorbent assay and western blot. We used the obtained antibody to detect endogenous MIP protein in hepatocellular carcinoma (HCC) cells and normal human hepatic cells L-O2 and found decreased MIP expression in HCC cells. The immunohistochemistry results further showed that the expression level of MIP protein in HCC tissues was lower than that in the corresponding adjacent non-cancerous tissues. The MIP protein was distributed mainly in the cytoplasm in HCC. In adjacent tissues, however, high expression of MIP was detected in the nucleus in addition to the cytoplasm. The prepared polyclonal antibody against MIP provides a useful tool for further research on the functions of MIP.

Down-regulation of metabolic proteins in hepatocellular carcinoma with portal vein thrombosis

BackgroundHepatocellular carcinoma is an aggressive malignancy with poor prognosis and easy to recur even the tumor is totally removed by surgery. Portal vascular invasion is one of the major factors contributing to tumor recurrence and poor prognosis. However, why hepatocellular carcinoma is easy to grow into vessels is unclear.MethodsSurgical specimens from seven hepatocellular carcinoma patients with portal vein thrombosis and seven patients without vascular invasion were utilized to analyze protein expression by proteomic technique. The proteins in the tumors were separated by 2-dimensional electrophoresis. Protein patterns in the gels were recorded as digitalized images. The differences of expression in hepatocellular carcinoma with or without portal vein thrombosis were identified by mass spectrometry.ResultsClinically, the tumors with portal vein thrombosis were larger than those without portal vein thrombosis. The median survival time for the patients with portal vein thrombosis was much shorter [4 (ranged 2.5–47) vs. 53 (ranged 33–85) months, p = 0.002]. By analyzing the protein expression in cancer tissues with or without portal vein thrombosis, the differences of protein expression were mainly metabolic enzymes. Carbonic anhydrase I, betaine–homocysteine S-methyltransferase 1, fumarate hydratase, isovaleryl-CoA dehydrogenase, short-chain specific acyl-CoA dehydrogenase and arginase-1 were all down-regulated in the tumors with portal vein thrombosis.ConclusionMetabolic enzymes and cytosol carbonic anhydrases were downregulated in hepatocellular carcinoma with portal vein thrombus. The deficiency of metabolic enzymes and cytosol carbonic anhydrases may alter cellular metabolisms and acid–base balance in hepatocellular carcinoma, which may facilitate to invade portal vein.

Oxaliplatin inhibits proliferation and migration of human hepatocellular carcinoma cells via GAS7C and the N-WASP/FAK/F-actin pathway.

The growth arrest-specific gene 7 (GAS7), a member of the growth-arrest-specific family, encodes three protein isoforms (GAS7A, GAS7B, and GAS7C) and plays a potential role in lung cancer as a tumor suppressor gene. In the present study, we found low endogenous expressions of GAS7C mRNA and protein in hepatocellular carcinoma (HCC) cell lines compared with normal liver cells, and that there was a distinct increase of GAS7C expression in HCC cells treated with oxaliplatin. CCK8, apoptosis, and Transwell migration assays showed that cell proliferation and motility of HepG2 and MHCC-97 H cells were inhibited by oxaliplatin, while apoptosis was increased. Interestingly, western blot analysis showed that treatment with oxaliplatin increased GAS7C and N-WASP protein levels and decreased the levels of proteins involved in the fibronectin/integrin/FAK pathway, such as FAK, in both HCC cell lines. In addition, ectopically overexpressed GAS7C obviously inhibited cell proliferation and cell motility. Flow cytometry results showed that overexpression of GAS7C induced apoptosis of HepG2 and MHCC-97 H cells. We further confirmed the correlation between GAS7C and the N-WASP/FAK/F-actin pathway by q-PCR and western blot analysis of in GAS7C-overexpressing HepG2 and MHCC-97 H cells. Inhibition of GAS7C substantially reversed the anti-cancer effect of oxaliplatin and blocked the activity of the N-WASP/FAK/F-actin pathway. Taken together, our results showed that oxaliplatin inhibits HCC cell proliferation and migration ability by up-regulating GAS7C and activating the N-WASP/FAK/F-actin pathway.

Granulin-epithelin precursor interacts with 78-kDa glucose-regulated protein in hepatocellular carcinoma

BackgroundGranulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach.MethodsThe membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections.ResultsWe identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman’s correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining.ConclusionsGRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.

Diagnostic utility of leptin and insulin-like growth factor binding protein-2 in hepatocellular carcinoma of diabetic and non-diabetic Egyptian patients

Purpose: To elucidate the possible diagnostic utility of adipokines and insulin growth factor binding proteins in hepatocellular carcinoma (HCC) diabetic subjects. Methods: Seventy five patients were divided equally into 3 groups as follows: healthy normal control (NC), non-diabetic hepatocellular carcinoma (HCC) and diabetic hepatocellular carcinoma (HCC-DM). Serum levels of leptin, insulin growth factor binding protein-2 (IGFBP-2) and alpha fetoprotein (AFP) were measured. Correlation and receiver operating characteristics (ROC) analysis was carried out. Results: HCC and HCC-DM groups showed changes in body mass index (BMI, p > 0.05 and p < 0.001 respectively), glucose, insulin, homeostatic model assessment-insulin resistance (HOMA-IR), liver function tests and AFP (p < 0.001). Leptin levels increased significantly in both HCC and HCC-DM (p < 0.001). Furthermore, IGFBP-2 showed significant increase in both groups (p < 0.001). Both leptin and insulin-like growth factor binding protein-2 (IGFBP-2) displayed significant positive correlation with AFP (p < 0.001). ROC analysis indicate different diagnostic accuracies for the tested markers for the various groups. Conclusion: Leptin and IGFBP-2 demonstrate significant potentials as diagnostic tools for HCC patients, especially diabetic cases, with IGFBP-2 displaying the highest diagnostic accuracy for HCC and HCC-DM groups. Keywords: Hepatocellular carcinoma, Diabetes mellitus, Leptin, Insulin-like growth factor-binding proteins-2, Adipokines

CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma.

AIMTo investigate the mechanism of chaperone-mediated autophagy (CMA)-induced resistance to irradiation-triggered apoptosis through regulation of the p53 protein in hepatocellular carcinoma (HCC).METHODSFirstly, we detected expression of lysosome-associated membrane protein 2a (Lamp-2a), which is the key protein of CMA, by western blot in HepG2 and SMMC7721 cells after irradiation. We further used shRNA Lamp-2a HCC cells to verify the radioresistance induced by CMA. Next, we detected the HMGB1 and p53 expression after irradiation by western blot, and we further used RNA interference and ethyl pyruvate (EP), as a HMGB1 inhibitor, to observe changes of p53 expression. Finally, an immunoprecipitation assay was conducted to explore the interaction between Lamp-2a and HMGB1, and the data were analyzed.RESULTSWe found the expression of Lamp-2a was increased on irradiation while apoptosis decreased in HepG2 and SMMC7721 cells. The apoptosis was increased markedly in the shRNA Lamp-2a HepG2 and SMMC7721 cells as detected by western blot and colony formation assay. Next, we found p53 expression was gradually reduced on irradiation but obviously increased in shRNA Lamp-2a cells. Furthermore, p53 increased the cell apoptosis on irradiation in Hep3B (p53-/-) cells. Finally, p53 levels were regulated by HMGB1 as measured through RNA interference and the EP treatment. HMGB1 was able to combine with Lamp-2a as seen by immunoprecipitation assay and was degraded via the CMA pathway. The decreased HMGB1 inhibited p53 expression induced by irradiation and further reduced the apoptosis in HCC cells.CONCLUSIONCMA pathway activation appears to down-regulate the susceptibility of HCC to irradiation by degrading HMGB1 with further impact on p53 expression. These findings have clinical relevance for radiotherapy of HCC.

Expression of β-catenin protein in hepatocellular carcinoma and its relationship with alpha-fetoprotein.

This study aimed to investigate the expression of β-catenin in hepatocellular carcinoma (HCC) tissues and its relationship with α-fetoprotein (AFP) in HCC. Immunohistochemistry was used to determine the expression of β-catenin in normal liver tissues (n=10), liver cirrhosis tissues (n=20), and primary HCC tissues (n=60). The relationship between β-catenin expression and clinical parameters of HCC was investigated. Real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of β-catenin in the liver cancer cell line SMMC-7721 transfected with a plasmid encoding AFP, and also the mRNA and protein expression levels of β-catenin were measured in the liver cancer cell line Huh7 before and after the transfection with AFP shRNA plasmids. The results showed that β-catenin was only expressed on the cell membrane in normal liver tissues. Its localization to the cytoplasm and nucleus of cells was observed in a small proportion of cirrhotic tissues or adjacent HCC tissues, and such ectopic expression of β-catenin was predominant in HCC tissues. The abnormal expression of β-catenin was correlated with serum AFP levels, cancer cell differentiation and vascular invasion (P<0.05). Additionally, the increased expression of AFP resulted in the upregulation of β-catenin mRNA and protein levels, while knockdown of AFP with AFP shRNA led to significantly decreased β-catenin mRNA and protein levels (P<0.05). It was suggested that the abnormal expression of β-catenin is implicated in hepatic carcinogenesis and development. AFP can lead to increased expression of β-catenin, which may account for the poor prognosis of AFP-associated HCC patients.

Expression of Scavenger receptor class A, member 5 protein in hepatocellular carcinoma and the relationship with promoter methylation

Objective To investigate the expression and clinical significance of Scavenger receptor class A, member 5 (SCARA5) in patients with hepatocellular carcinoma (HCC), and to explore the relationship between methylation status of SCARA5 gene promoter and its protein expression. Methods SCARA5 expression in tumor and matched adjacent non-cancerous liver tissues from 112 patients with HCC was deteced by immunohistochemistry using tissue microarray technology. The relationship between the protein expression of SCARA5 and the clinical, pathological features in patients with HCC was also analyzed. The promoter methylation patterns of SCARA5 gene in tumor and matched adjacent non-cancerous liver tissues were also measured by methylation-specific PCR technique. Results Negative and weak staining of SCARA5 was observed in 77.7% (87/112) of the HCC tissue samples, contrast to 25.0% (28/112) in the adjacent non-cancerous liver tissue samples. SCARA5 was significantly decreased in HCC tissues (P 0.05). However, the expression of SCARA5 was significantly correlated with tumor size, vascular invasion, Edmondson-Steiner grade and TNM stage (P<0.05). Methylation of SCARA5 gene promoter was found in 60.7% of the all tumor tissue samples. However, methylation was only found in 11.6% of the adjacent non-tumor samples, which was significantly lower than that in tumor tissue samples (P<0.05). Meanwhile, the expression of SCARA5 in tumor tissue samples was negatively correlated with promoter methylation status (P<0.05). Conclusion It suggests that the decreased expression of SCARA5 may have an important role in the development of HCC, and the promoter methylation of SCARA5 gene may be one of the reasons for the decreased expression of SCARA5. Key words: Carcinoma, hepatocellular; Scavenger receptor class A, member 5; Tumor suppressor gene; Methylation

Expression Pattern and Clinical Significance of Gab2 Protein in Hepatocellular Carcinoma

To investigate the expression pattern of Gab2 in hepatocellular carcinoma (HCC) and explore the correlation between Gab2 expression and clinicopathological features of HCC patients. The prognostic significance of Gab2 expression is evaluated to determine the possible role in the progression of HCC. Gab2 expression was detected by immunohistochemistry in 90 HCC samples and matched adjacent noncancerous liver tissues. The mRNA and protein of Gab2 in HCC and normal liver cell lines were examined by quantitative RT-PCR and western blot, respectively. The correlation between Gab2 expression of tumor tissues and clinicopathological parameters was analyzed by Chi-squared test or Fisher's exact test. The association between Gab2 expression and overall survival percentage after surgery was evaluated by Kaplan-Meier method. Gab2 expression was elevated in HCC tissues compared with matched normal liver tissues (p < 0.001). Gab2 was upregulated in a subset of HCC cell lines. Among the clinical and pathological features, Gab2 expression was correlated to the histologic grade of HCC tissues (p < 0.05). Kaplan-Meier analysis of the cumulative survival rate after surgery indicated that no statistical difference existed between high-Gab2 and low-Gab2 expression group (p = 0.8297). Gab2 may be involved in the onset and progression of HCC. Gab2 expression is unable to serve as an independent prognosis factor in HCC patients.

Yes-Associated Protein in Hepatocellular Carcinoma is Associated with Tumor Differentiation and Patient Age at Diagnosis, but not Markers of HBV Infection.

It was necessary to assess the relationship between Yes-associated protein (YAP) and some clinical features of hepatocellular carcinoma (HCC), especially hepatitis B virus (HBV) correlation factors as they relate to tumorigenesis. A tissue microarray including 84 HCC samples was retrospectively analyzed by immunohistochemistry. This study showed that YAP expression was associated with HCC differentiation and the patient age at diagnosis of HCC. The mean age at diagnosis of YAP(+) HCC patients was 46.19 ± 9.45 years old, which is youn- ger than 51.40 ± 12.51 years old found for YAP(-) HCC patients (< 0.048). There was no significant correlation between YAP expression and HBV correlation factors (HBsAg, HBV DNA, and the duration of hepatitis B infec- tion). YAP(+) HCC patients had a younger mean age at diagnosis and more poor-differentiation charac- teristics of HCC. However, there were no independent HBV correlation factors.

A study on the structural features of SELK, an over-expressed protein in hepatocellular carcinoma, by molecular dynamics simulations in a lipid-water system.

Human SELK is a small trans-membrane selenoprotein characterized by a single trans-membrane helix, while the N-terminal region protrudes into the lumen and the long C-terminal domain into the cytoplasm. SELK is over-expressed in some cancers, like hepatocellular carcinoma; however its precise role in cancer development is presently unknown. SELK is involved in promoting the calcium flux, catalyzing palmitoylation reactions and protein degradation in the endoplasmic reticulum (ER). Therefore, this protein should bind many different proteins like p97/VCP in the supramolecular complex involved in the ER degradation pathway. To study the structural features of SELK in the membrane, we have modeled the protein and then subjected it to molecular dynamics simulations in a lipid-water system. The model shows a N-terminal domain with three β-strands and a short helix, a well-defined trans-membrane helix and a C-terminal domain that lacks a persistent secondary structure and contains long disordered regions. The trajectory analysis during the simulation evidences that: (i) the N-terminal region explores a limited conformational space and is stabilized by intra-peptide H-bonds as well with membrane lipids and water, (ii) the trans-membrane helix was found to be quite stable and (iii) the disordered C-terminal region is stabilized by H-bonds with clustered water molecules as well as by rapidly interchanging intra-peptidic H-bonds, with a structural tendency to compact around the four HUB residues found for this domain. Moreover, N-terminal and C-terminal clusters are distributed differently in the conformational space suggesting that their dynamics are coupled complicatedly through the membrane. Further analyses have shown that the N-terminal has a tendency to pivot around the insertion with the TM-helix through the fluctuations of the three β-strands, which, in turn, show features similar to WW-domains. These results will be useful to study the SELK, SELS and VCP complex representing an interesting druggable target for cancer.

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.

Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma.

Cell division cycle 5-like (Cdc5L), as a pre-mRNA splicing factor, is a regulator of mitotic progression. Previous study found that deletion of endogenous Cdc5L decreases the cell viability via dramatic mitotic arrest, while the role of Cdc5L in cancer biology remains under debate. To investigate the involvement of Cdc5L in the progression of hepatocellular carcinoma (HCC). In this study, the expression of Cdc5L was evaluated by Western blot in 8 paired fresh HCC tissues and immunohistochemistry on 116 paraffin-embedded slices. We treated HCC cells by nocodazole to analyze the role of Cdc5L in mitotic progress. To determine whether Cdc5L could regulate the proliferation of HCC cells, we increased endogenous Cdc5L and analyzed the proliferation of HCC cells using Western blot, CCK8, flow cytometry assays, and colony formation analyses. Furthermore, Cdc5L-siRNA oligos were used to confirm that Cdc5L plays an essential role in HCC development. Cdc5L was highly expressed in HCC and significantly associated with multiple clinicopathological factors, including AJCC stage, tumor size, and Ki-67. Besides, univariate and multivariate survival analyses demonstrated that high Cdc5L expression was an independent prognostic factor for HCC patients' poor survival. Overexpression of Cdc5L favors cell cycle progress of HCC cells, while downregulation of Cdc5L results in cell cycle arrest at G2/M phase and reduced cell proliferation of HCC cells. Our findings suggested that Cdc5L could play an important role in the tumorigenesis of HCC and thus be a potential therapeutical target to prevent HCC progression.

Mass-Selected Site-Specific Core-Fucosylation of Serum Proteins in Hepatocellular Carcinoma

A mass spectrometry-based methodology has been developed to screen for changes in site-specific core-fucosylation (CF) of serum proteins in early stage HCC with different etiologies. The methods involve depletion of high-abundance proteins, trypsin digestion of medium-to-low-abundance proteins into peptides, iTRAQ labeling, and Lens culinaris Agglutinin (LCA) enrichment of CF peptides, followed by endoglycosidase F3 digestion before mass spectrometry analysis. 1300 CF peptides from 613 CF proteins were identified from patients sera, where 20 CF peptides were differentially expressed in alcohol (ALC)-related HCC samples compared with ALC-related cirrhosis samples and 26 CF peptides changed in hepatitis C virus (HCV)-related HCC samples compared with HCV-related cirrhosis samples. Among these, we found three CF peptides from fibronectin upregulated in ALC-related HCC samples compared with ALC-related cirrhosis samples with an AUC (area under the curve) value of 0.89 at site 1007 with a specificity of 85.7% at a sensitivity of 92.9% (generated with 10-fold cross-validation). When combined with the AFP index, the AUC value reached to 0.92 with a specificity of 92.9% at a sensitivity of 100%, significantly improved compared to that with AFP alone (LR test p < 0.001). In HCV-related samples, the CF level of cadherin-5 at site 61 showed the best AUC value of 0.75 but was not as promising as that of fibronectin site 1007 for ALC-related samples. The CF peptides of fibronectin may serve as potential biomarkers for early stage HCC screening in ALC-related cirrhosis patients.

Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential.

Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP-like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.

Regulation of MET receptor tyrosine kinase signaling by suppressor of cytokine signaling 1 in hepatocellular carcinoma.

Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.

Hepatocellular carcinoma: immunohistochemical characteristics of apoptosis and cell proliferation activity.

Background. Hepatocellular carcinoma which takes about 85% of primary liver cancer, is the most aggressive and prognostically unfavorable tumor. Current information about the level of apoptosis and cell proliferative activity of HCC are contradictory. Objective. To determine the apoptotic and proliferative activity of hepatocellular carcinoma of the liver by the immunohistochemically level of p53, caspase-3 and Ki-67 expression. Methods. Trephine biopsies of 53 patients with hepatocellular carcinoma were studied. Results. It was found that in 62.26% of cases the hyperexpression of nuclear p53 protein in cells was detected, in 20.75% of cases - a high level of expression of this protein, and in 16.98% of cases - a low level of p53 expression. The average area of p53-immunopositive cells was 52,07±33,97%. Simultaneously, in 47.16% of hepatocellular carcinoma it was detected a weak level of cytoplasmic expression of caspase-3, in 26.42% of the patients moderate level of expression of caspase-3 by hepatocellular carcinoma cells was determined, pronounced expression of caspase-3 was detected in hepatocellular carcinoma cells 26, 42%. Caspase-3-immunopositive cells occupied 48,21±27,66% of the total area of hepatocellular carcinoma. It is established that in the hepatocellular carcinoma prevailed moderate level of expression of Ki-67, which was observed in 66.04% of patients, in 22.64% it was high and in 11.32% of cases – it was low. Immupositive Ki-67 cells occupied 53,39±23,25% of the total area of the tumor. Conclusion. Hyperexpression of nuclear p53 protein in hepatocellular carcinoma was associated with weak and low level of apoptosis of tumor cells and moderate level of their proliferation.

Increased expression of CCN2, epithelial membrane antigen, and fibroblast activation protein in hepatocellular carcinoma with fibrous stroma showing aggressive behavior.

Tumor behavior is affected by the tumor microenvironment, composed of cancer-associated fibroblasts (CAFs). Meanwhile, hepatocellular carcinomas (HCC) with fibrous stroma reportedly exhibit aggressive behavior suggestive of tumor-stroma interaction. However, evidence of the crosstalk remains unclear. In this study, CCN2, epithelial membrane antigen (EMA), fibroblast activation protein (FAP), and keratin 19 (K19) expression was studied in 314 HCCs (cohort 1), 42 scirrhous HCCs (cohort 2), and 36 chronic hepatitis/cirrhosis specimens by immunohistochemistry. Clinicopathological parameters were analyzed according to the expressions of these markers. In tumor epithelial cells from cohort 1, CCN2 and EMA were expressed in 15.3% and 17.2%, respectively, and their expressions were more frequent in HCCs with fibrous stroma (≥5% of tumor area) than those without (P<0.05 for all); CCN2 expression was well correlated with K19 and EMA expression. In tumor stromal cells, FAP expression was found in 6.7%. In cohort 2, CCN2, EMA, and FAP expression was noted in 40.5%, 40.5%, and 66.7%, respectively, which was more frequent than that in cohort 1 (P<0.05 for all). Additionally, EMA expression was associated with the expression of K19, CCN2, and FAP (P<0.05 for all); EMA expressing tumor epithelial cells showed a topographic closeness to FAP-expressing CAFs. Analysis of disease-free survival revealed CCN2 expression to be a worse prognostic factor in both cohort 1 (P = 0.005) and cohort 2 (P = 0.023), as well as EMA as a worse prognostic factor in cohort 2 (P = 0.048). In conclusion, expression of CCN2, EMA, and FAP may be involved in the activation of CAFs in HCC, giving rise to aggressive behavior. Significant correlation between EMA-expressing tumor cells and FAP-expressing CAFs and their topographic closeness suggests possible cross-talk between tumor epithelial cells and stromal cells in the tumor microenvironment of HCC.

Vitamin B12 and its binding proteins in hepatocellular carcinoma and chronic liver diseases

Background. The vitamin B12 (B12)-binding protein haptocorrin (HC) has proven to be a potentially useful biomarker in patients with fibrolamellar hepatocellular carcinoma (HCC). Little is known concerning the level of HC and other B12-related proteins in patients with HCC as compared to patients with other chronic liver diseases (CLDs) and healthy controls. We hypothesized that HC could be a biomarker of HCC. Aims. To investigate levels of HC and B12-related proteins in HCC compared to CLDs and healthy controls. Methods. We investigated two patient populations: A cross-sectional cohort of HCC patients (n = 130), CLD patients (n = 102) and healthy controls (n = 46) and a cohort of 38 HCC patients studied at baseline and 1, 4, and 12 weeks following ablative treatment. Patients were evaluated by standard biochemistry, Child–Pugh-score and Barcelona Clinic Liver Cancer (BCLC) classification. We analyzed total B12 by routine methods and HC, transcobalamin (TC), B12 saturated TC (holoTC), and the soluble cell surface receptor for holoTC (sCD320) by in-house enzyme-linked immunosorbent assay. Results. HC showed higher median (range) levels for both HCC (590 [290–5860]) and CLD patients (620 [310–4010]) compared to controls (460 [250–2020]) (p < 0.01). Total B12, TC, holoTC, and sCD320 showed elevated levels in both HCC and CLD compared to controls. Only holoTC changed following treatment, without a concurrent change in TC. Conclusion. B12 and B12-related proteins (total B12, HC, TC, holoTC, and sCD320) show elevations in both HCC and CLD patients compared to controls, suggesting a relation to CLD in general rather than to primary liver cancer. Thus, HC is not useful as a biomarker for HCC.

Long Noncoding RNAs in Interaction With RNA Binding Proteins in Hepatocellular Carcinoma.

Background:Gene expression microarrays' analyses provide a description of long noncoding RNAs (lncRNAs) with lack of coding protein function that is often important in human cancer.Objectives:A number of lncRNAs that have been well characterized in hepatocellular carcinoma (HCC) have been scheduled in this study to discuss for protein–lncRNA interaction.Materials and Methods:The identified lncRNAs were analyzed by bioinformatics tools, starBase and lncRNA db, to anticipate the RNA-binding proteins (RBPs) that tend to interact to HCC-related lncRNAs. The most important predicted RBPs in interaction with well-known lncRNAs in HCC were briefly discussed.Results:The lncRNAs HOTTIP, H19, HOTAIR, MALAT1, antisense Igf2r (AIR), HOXA13, GTL2 (also called MEG3) and uc002mb have been reported in association with HCC. Besides, this study predicted that eIF4AIII, PTB and FUS were the most involved RBPs in interaction with HCC-related lncRNAs.Conclusions:This information provides an explanation for the previously valuable literature on the functions of lncRNAs and suggest for the novel therapeutic targeting.