Decreased Expression of TMEM173 Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal cancer types, and chronic infection with Hepatitis B Virus (HBV) is identified as the strongest risk factor for HCC. Transmembrane Protein 173 (TMEM173) is a pattern recognition receptor which functions as a major regulator of the innate immune response to viral and bacterial infections. However, the prognostic value of TMEM173 in HCC remains elusive. Thus, we aimed to evaluate the potential prognostic significance of TMEM173 expression in HCC patients following curative resection. Immunohistochemistry was used to detect TMEM173 expression in 96 HCC patients. We found that TMEM173 protein expression was remarkably decreased in tumor tissues compared to non-tumor tissues, and that TMEM173 staining intensity was inversely correlated with tumor size, tumor invasion TNM stage and overall survival (OS) in HCC patients. Multivariate analysis supported TMEM173 as an independent prognostic factor, and identified that combining TMEM173 expression with TNM stage showed better prognostic efficiency for OS in HCC patients. In summary, TMEM173 was discovered having an independent prognostic value and may serve as a potential immunotherapeutic target for HCC.     

Low SIRT3 Expression Correlates with Poor Differentiation and Unfavorable Prognosis in Primary Hepatocellular Carcinoma

SIRT3, a mitochondrial sirtuin belonging to nicotinamide adenine nucleotide (NAD) dependent deacetylases, is implicated in metabolism, longevity and carcinogenesis. SIRT3 expression and its significance in hepatocellular carcinoma (HCC) remain largely unclear. In this study, we demonstrated that SIRT3 expression in HCC tissue was much lower than that in paracarcinoma tissue, at both mRNA and protein levels. The cutoff value for low SIRT3 expression in HCC was defined according to receiver operating characteristic curve (ROC) analysis. As disclosed by immunohistochemistry (IHC) results, low SIRT3 expression was present in 67.3% (167/248) of HCC cases. Furthermore, low expression of SIRT3 was significantly correlated to differentiation (P = 0.013), clinical stage (P = 0.005), serum AFP level (P<0.01), tumor multiplicity (P = 0.026) and relapse (P = 0.028). Moreover, Kaplan-Meier analysis indicated that low SIRT3 expression associated with unfavorable overall survival (P<0.01) and recurrence-free survival (P = 0.004). The prognostic impact of SIRT3 was further confirmed by stratified survival analysis. Importantly, multivariate analysis revealed that low SIRT3 expression was an independent poor prognostic marker for overall survival (Hazard Ratio (HR) 0.555, 95% confidence interval (95% CI) 0.344–0.897, P = 0.016). Collectively, we conclude that SIRT3 is decreased in HCC and is a novel unfavorable marker for prognosis of patients with this fatal disease.     

Numb Promotes Cell Proliferation and Correlates with Poor Prognosis in Hepatocellular Carcinoma

Background

Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, cell adhesion, cell migration and a number of signaling pathways, but evidence for a substantial involvement of Numb in HCC has remained unclear. The present study was aimed to investigate the clinical and prognostic significance of Numb and its role in hepatocellular carcinoma (HCC).

Methodology

The expression of Numb was detected in 107 cases of clinical paraffin-embedded hepatocellular carcinoma tissues,5 matched paris of fresh tissues and six hepatocellular cell lines by immunohistochemistry with clinicopathological analyses,RT-PCR or Western blot. Moreover, loss of function and gain of function assays were performed to evaluate the effect of Numb on cell proliferation in vitro.

Conclusions

We found that Numb was obviously up-regulated in HCC tissues and cell lines (p<0.05). The Numb up-regulation correlated significantly with poor prognosis, and Numb status was identified as an independent prognostic factor. Over-expression of Numb increased proliferation in SMMC-7721 and BEL-7402 cells, while knock-down of Numb showed the opposite effect. Our study indicates that Numb up-regulation significantly correlates with cell proliferation and poor prognosis in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy in hepatocellular carcinoma treatment.     

Expression of Hypoxic Marker Carbonic Anhydrase IX Predicts Poor Prognosis in Resectable Hepatocellular Carcinoma

Carbonic anhydrase IX (CA-IX), a hypoxia marker, correlates with tumor progression in a variety of human cancers. However, the role of CA-IX in hepatocellular carcinomas (HCCs) remains largely unknown. We examined the expression of 277 unifocal, resectable, primary HCC tumors using immunohistochemistry. The CA-IX protein was expressed in 110 of the 227 (48.5%) HCC tumors. The expression of CA-IX correlated with younger age (P = 0.0446), female sex (P = 0.0049), high serum α-fetoprotein levels (P<1x10^-6), larger tumor size (P = 0.0031), high tumor grade P<1x10^-6) and high tumor stage (P = 1.5x10^-6). Patients with HCC tumors that expressed CA-IX were more likely to have lower 5-year disease-free survival (DFS; P = 0.0001) and 5-year overall survival (OS; P<1x10^-6). The multivariate analysis indicated that CA-IX expression was an independent predictor for high tumor stage (P = 0.0047) and DFS (P = 0.0456), and a borderline predictor for OS (P = 0.0762). Furthermore, CA-IX expression predicted poor DFS and OS in patients with high tumor stage (P = 0.0004 and P<1x10^-6, respectively). Interestingly, CA-IX expression might contribute to the worse prognosis of female patients with advanced HCCs. Our study indicates the expression of the CA-IX protein is a crucial predictor of poor prognosis in resectable HCC, and it is also an unfavorable prognostic predictor in HCC patients with high tumor stage.     

Increased Expression of CAP2 Indicates Poor Prognosis in Hepatocellular Carcinoma

CAP2 has been suggested as a potential diagnostic biomarker for early hepatocellular carcinoma (HCC). However, its prognostic significance in HCC remains unclear. Here, we show that CAP2 expression is much higher in HCC tissues than that in paracarcinoma tissues, at both mRNA and protein levels. Data of immunohistochemistry (IHC) revealed that CAP2 was markedly up-regulated in 77.3% of HCC cases. High CAP2 expression, defined by the median score of IHC, was present in 53.3% of the patients. Kaplan-Meier analysis indicated that high CAP2 expression was associated with poor overall survival (P < .0001), disease-free survival (P = .013) and recurrence probability (P = .004) in a training cohort of 312 HCC patients. The prognostic implication of CAP2 in HCC was further confirmed in a validation cohort of 208 HCC patients and by stratified survival analysis. Multiple Cox regression analysis indicated CAP2 as an independent predictor for overall survival (hazard ratio (HR) = 1.615, 95% confidence interval: 1.345-1.938, P < .001). Collectively, we conclude that CAP2 is increased in HCC and is a novel unfavorable biomarker for prognostic prediction for patients with this deadly disease.     

Decreased Expression of Beclin 1 Correlates Closely with Bcl-xL Expression and Poor Prognosis of Ovarian Carcinoma

Background

It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.

Methodology/Principal Findings

In the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P<0.05). In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (P<0.01), and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (P = 0.013). In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.

Conclusions/Significance

Our findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.     

High PD-L1 Expression Correlates with Metastasis and Poor Prognosis in Oral Squamous Cell Carcinoma

PD-L1 has been widely demonstrated to contribute to failed antitumor immunity. Blockade of PD-L1 with monoclonal antibody could modulate the tumor immune environment to augment immunotherapy. PD-L1 expression is also detected in several types of cancer and is associated with poor prognosis. However, the prognostic role of PD-L1 in oral squamous cell carcinoma (OSCC) is still controversial. Our aim was to determine the role of PD-L1 in the prognosis of OSCC patients to identify its potential therapeutic relevance. PD-L1 immunoreactivity was analyzed by immunohistochemistry in 305 cancer specimens from primary OSCC patients. The medium follow-up time after surgery was 3.8 years (range from 0.1 to 11.1 years). The prognostic value of PD-L1 on overall survival was determined by Kaplan-Meier analysis and Cox proportional hazard models. Higher PD-L1 expression is more likely in tumor tissues of female than male OSCC patients (P = 0.0062). Patients with distant metastasis also had high PD-L1 expression (P = 0.0103). Multivariate analysis identified high PD-L1 expression as an independent risk factor in males and smokers (males: hazard ratio = 1.556, P = 0.0077; smokers: hazard ratio = 2.058, P = 0.0004). We suggest that PD-L1 expression, determined by IHC staining, could be an independent prognostic marker for OSCC patients who are male or who have a smoking habit.     

High Expression of FLOT1 Is Associated with Progression and Poor Prognosis in Hepatocellular Carcinoma

Background

The flotillin family member flotillin-1 (FLOT1) encodes a caveolae-associated, integral membrane protein that belongs to lipid raft family and involves in vesicular trafficking and signal transduction. However, the role of FLOT1 in development and progression of cancer remains largely unknown. The present study was aimed to investigate the clinical and prognostic significance of FLOT1 in hepatocellular carcinoma (HCC).

Methods

Real-time PCR and western blot analyses were applied to examine FLOT1 expression in fourteen HCC cell lines and one normal hepatic cell line, ten pairs of primary HCC and matched adjacent noncancerous liver tissues from the same patient. Immunohistochemistry (IHC) was performed to examine FLOT1 protein expression in paraffin-embedded tissues from 196 HCC patients. Statistical analyses were applied to evaluate the diagnostic value and associations of FLOT1 expression with clinical parameters.

Results

FLOT1 expression was evidently up-regulated in HCC tissues compared with that in the matched adjacent noncancerous liver tissues. In the 196 cases of tested HCC samples, FLOT1 protein level was positively correlated with Tumor size (P = 0.025), clinical stage (P<0.002), CLIP stage (P<0.001), vascular invasion (P<0.001), relapse (P<0.001), and serum AFP levels (P = 0.025). Patients with higher FLOT1 expression had shorter overall survival time, whereas those with lower FLOT1 expression had longer survival time.

Conclusions

Our study demonstrated FLOT1 is associated with aggressive characteristics of HCC, and suggested the possibility of its use as a prognostic marker in patients with HCC.     

Decreased Expression of PTPN12 Correlates with Tumor Recurrence and Poor Survival of Patients with Hepatocellular Carcinoma

Background

Protein tyrosine phosphatase non-receptor type 12 (PTPN12), has been identified as a potent tumor suppressor in human cancers and a critical regulator of cell adhesion and migration. However, the PTPN12 expression and its prognostic significance in HCC have not been well elucidated.

Methodology/Principal Findings

In this study, tissue microarray-based immunohistochemistry (IHC) was investigated in an HCC cohort with adjacent liver tissues as controls. The resulting data were analyzed using receiver operating characteristic curves, Spearman''s rank correlation, Kaplan-Meier plots and Cox proportional hazards regression modeling. Our results showed that decreased expression of PTPN12 was more frequently observed in HCC tissues compared to the adjacent non-tumorous liver tissues. Further correlation analyses indicated that the decreased PTPN12 expression was closely correlated with tumor recurrence (P = 0.015). Univariate analysis showed a significant association between decreased expression of PTPN12 and adverse cancer-specific survival and recurrence-free survival (P<0.001). In different subsets of overall patients, PTPN12 expression was also a prognostic indicator in patients with stage I/II or stage III/IV (P<0.05). Importantly, multivariate analysis (P<0.05) identified PTPN12 expression in HCC as an independent prognostic factor.

Conclusions/Significance

Our findings provide a basis for the concept that PTPN12 protein expression is frequently decreased or lost in human HCC tissues and that decreased PTPN12 expression may represent an acquired recurrence phenotype of HCC and that PTPN12 expression may act as a biomarker of prognosis for patients with HCC.     

Decrease of Fibulin-3 in Hepatocellular Carcinoma Indicates Poor Prognosis

Fibulin-3, originally identified in senescent and Werner syndrome fibroblasts, has been implicated in cell morphology, growth, adhesion and motility. Fibulin-3 exhibits both antitumor and oncogenic activities towards human cancers; however, the role of Fibulin-3 in hepatocellular carcinoma (HCC) remains elusive. In this study, we showed that both the mRNA and protein levels of Fibulin-3 were remarkably downregulated in HCC cell lines and fresh tissues. Immunohistochemical data revealed that Fibulin-3 was decreased in tumorous tissues in 67.1% (171/255) of cases compared to the corresponding adjacent nontumorous tissues. The results of statistical analysis indicated that low Fibulin-3 expression, defined by the receiver operating characteristic curve (ROC), was significantly associated with tumor differentiation (P = 0.008), clinical stage (P = 0.014) and serum AFP levels (P<0.01). Furthermore, Kaplan-Meier and multivariate analysis suggested that Fibulin-3 is an independent negative prognostic indicator for both overall (P<0.001) and recurrence-free (P = 0.036) survival. In addition, an in vitro study demonstrated that knockdown of Fibulin-3 by siRNA markedly increased cell viability and promoted cell invasion in HCC cells. Collectively, our data suggest that Fibulin-3 exhibits antitumor effects towards HCC and serves as a biomarker of unfavorable prognosis for this deadly disease.     

A Seven-microRNA Expression Signature Predicts Survival in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth common cancer. The differential expression of microRNAs (miRNAs) has been associated with the prognosis of various cancers. However, limited information is available regarding genome-wide miRNA expression profiles in HCC to generate a tumor-specific miRNA signature of prognostic values. In this study, the miRNA profiles in 327 HCC patients, including 327 tumor and 43 adjacent non-tumor tissues, from The Cancer Genome Atlas (TCGA) Liver hepatocellular carcinoma (LIHC) were analyzed. The associations of the differentially expressed miRNAs with patient survival and other clinical characteristics were examined with t-test and Cox proportional regression model. Finally, a tumor-specific miRNA signature was generated and examined with Kaplan–Meier survival, univariate\multivariate Cox regression analyses and KEGG pathway analysis. Results showed that a total of 207 miRNAs were found differentially expressed between tumor and adjacent non-tumor HCC tissues. 78 of them were also discriminatively expressed with gender, race, tumor grade and AJCC tumor stage. Seven miRNAs were significantly associated with survival (P value <0.001). Among the seven significant miRNAs, six (hsa-mir-326, hsa-mir-3677, hsa-mir-511-1, hsa-mir-511-2, hsa-mir-9-1, and hsa-mir-9-2) were negatively associated with overall survival (OS), while the remaining one (hsa-mir-30d) was positively correlated. A tumor-specific 7-miRNAs signature was generated and validated as an independent prognostic predictor. Collectively, we have identified and validated an independent prognostic model based on the expression of seven miRNAs, which can be used to assess patients’ survival. Additional work is needed to translate our model into clinical practice.     

Embryonic Morphogen Nodal Is Associated with Progression and Poor Prognosis of Hepatocellular Carcinoma

Background

Nodal, a TGF-β-related embryonic morphogen, is involved in multiple biologic processes. However, the expression of Nodal in hepatocellular carcinoma (HCC) and its correlation with tumor angiogenesis, epithelial-mesenchymal transition, and prognosis is unclear.

Methods

We used real-time PCR and Western blotting to investigate Nodal expression in 6 HCC cell lines and 1 normal liver cell line, 16 pairs of tumor and corresponding paracarcinomatous tissues from HCC patients. Immunohistochemistry was performed to examine Nodal expression in HCC and corresponding paracarcinomatous tissues from 96 patients. CD34 and Vimentin were only examined in HCC tissues of patients mentioned above. Nodal gene was silenced by shRNA in MHCC97H and HCCLM3 cell lines, and cell migration and invasion were detected. Statistical analyses were applied to evaluate the prognostic value and associations of Nodal expression with clinical parameters.

Results

Nodal expression was detected in HCC cell lines with high metastatic potential alone. Nodal expression is up-regulated in HCC tissues compared with paracarcinomatous and normal liver tissues. Nodal protein was expressed in 70 of the 96 (72.9%) HCC tumors, and was associated with vascular invasion (P = 0.000), status of metastasis (P = 0.004), AFP (P = 0.049), ICGR₁₅ (indocyanine green retention rate at 15 min) (P = 0.010) and tumor size (P = 0.000). High Nodal expression was positively correlated with high MVD (microvessal density) (P = 0.006), but not with Vimentin expression (P = 0.053). Significantly fewer migrated and invaded cells were seen in shRNA group compared with blank group and negative control group (P<0.05). High Nodal expression was found to be an independent factor for predicting overall survival of HCC.

Conclusions

Our study demonstrated that Nodal expression is associated with aggressive characteristics of HCC. Its aberrant expression may be a predictive factor of unfavorable prognosis for HCC after surgery.     

Methylation Profiles Reveal Distinct Subgroup of Hepatocellular Carcinoma Patients with Poor Prognosis

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.