Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.     

Serum microRNAs as biomarkers for hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection

Background

MicroRNAs (miRNAs) have been shown to anticipate great cancer diagnostic potential. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for hepatocellular carcinoma (HCC).

Methodology/Principal Findings

This study was divided into four phases: (I) Ten candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 10 HCC patients with chronic hepatitis B virus (HBV) infection and 10 age- and sex-matched healthy subjects. (II) Marker validation by real-time RT-PCR on HBV patients with (n = 48) or without HCC (n = 48), and healthy subjects (n = 24). (III) Marker detection by real-time RT-PCR in sera from another 14 HCC patients before and 1 month after surgical resection. (IV) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of HCC patients. Although miR-222, miR-223 or miR-21 were significantly up- or down-regulated between HCC patients and healthy controls, no significant difference was observed in the levels of these miRNAs between HBV patients without and with HCC. MiR-122 in serum was significantly higher in HCC patients than healthy controls (p<0.001). More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples. Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line (p = 0.043).

Conclusions/Significance

Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for detection of HCC in healthy subjects, moreover, it might serve as a novel biomarker for liver injury but not specifically for detection of HCC in chronic HBV infection patients.     

Circulating microRNAs,miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC

The performance of circulating biomarkers for the diagnosis of hepatocellular carcinoma (HCC) is sub-optimal. In this study we tested circulating microRNAs as biomarkers for HCC in cirrhotic patients by performing a two stage study: a discovery phase conducted by microarray and a validation phase performed by qRT-PCR in an independent series of 118 patients. Beside miRNAs emerged from the discovery phase, miR-21, miR-221, miR-519d were also tested in the validation setting on the basis of literary and tissue findings. Deregulated microRNAs were assayed in HCC-derived cells in the intracellular compartment, cell culture supernatant and exosomal fraction. Serum and tissue microRNA levels were compared in 14 patients surgically treated for HCC. From the discovery study, it emerged that seven circulating microRNAs were differentially expressed in cirrhotic patients with and without HCC. In the validation set, miR-939, miR-595 and miR-519d were shown to differentiate cirrhotic patients with and without HCC. MiR-939 and miR-595 are independent factors for HCC. ROC curves of miR-939, miR-595 and miR-519d displayed that AUC was higher than AFP. An exosomal secretion of miR-519d, miR-21, miR-221 and miR-1228 and a correlation between circulating and tissue levels of miR-519d, miR-494 and miR-21 were found in HCC patients. Therefore, we show that circulating microRNAs deserve attention as non-invasive biomarkers in the diagnostic setting of HCC and that exosomal secretion contributes to discharging a subset of microRNAs into the extracellular compartment.     

Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase Ⅱ in Hepatocellular Carcinoma

Early detection and intervention are key strategies to reduce mortality, increase longterm survival, and improve the therapeutic effects of hepatocellular carcinoma(HCC) patients.Herein, the isobaric tag for relative and absolute quantitation(iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues,surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II(CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.     

MicroRNA signature in hepatocellular carcinoma patients: identification of potential markers

MicroRNAs (miRNAs) play important roles in liver pathologies and they are potential biomarkers for diagnosis of liver diseases progression. Changes in miRNA sera expression can be used as non-invasive biomarkers for hepatocellular carcinoma (HCC). The aim of the study was to identify the miRNome profiling of HCC and its diagnostic value in distinguishing HCC from healthy individuals. Expression profiles of miRNAs in serum samples of 20 HCC patients and 10 healthy controls were detected. Whole miRNome profiling was done using next generation sequencing. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of the deregulated miRNAs for discriminating HCC cases from healthy controls. MiRNA 142 was highly expressed in HCC (P value?=?0.023) while miRNAs 191, 22, and 126 were higher in the controls (P value?=?0.005, 0.034, 0.010 respectively). We have identified 5 novel miRNAs and they were highly expressed in HCC than controls. Analysis of ROC curve demonstrated that these deregulated miRNAs can be used as a reliable biomarker for detection of HCC with high diagnostic accuracy (AUC?=?0.93). We have detected a panel of serum miRNAs that can be used as a reliable noninvasive screening biomarker of HCC. The study recommends further research to shed light on a possible role of the newly discovered novel miRNAs in HCC pathogenesis.

     

Serum miR-128-2 Serves as a Prognostic Marker for Patients with Hepatocellular Carcinoma

Circulating miRNAs are promising biomarkers for predicting the aggressiveness of hepatocellular carcinoma (HCC). We aimed to identify differentially expressed miRNAs in the serum of HCC patients with different Barcelona Clinic Liver Cancer (BCLC) stage, and to investigate the potential of serum miRNAs as biomarkers for patient outcomes. In the discovery stage, TaqMan Low-Density Array was used to test the difference in levels of serum miRNAs between 20 patients with portal vein tumor thrombosis (PVTT) and 20 patients without PVTT. The detected serum miRNAs then were validated in 182 patients. Fifteen serum miRNAs showed more than two-fold higher expression in patients with PVTT, and miR-128-2 was found to be significantly up-regulated and was selected for further validation. In the validation stage, patients were divided into two groups with low or high serum miR-128-2 using the median expression level of all 182 cases as the cut-off point. Kaplan-Meier analysis revealed that patients with low level of serum miR-128-2 had favorable trends of survival (log rank = 13.031, p < 0.001). The median survivals for patients with a low and high level of serum miR-128-2 were 625 (95% CI, 527–722) days and 426 (95% CI, 362–491) days, respectively. MiR-128-2 was also an independent factor of overall survival (p = 0.001, HR 2.793, 95%CI 1.550, 5.033). Serum levels of the ubiquitously expressed miR-128-2 showed no significant correlation with parameters of liver damage or liver function. In addition, expressions of miR-128-2 in HCC tissues were up-regulated in comparison with adjacent non-tumor tissues. In conclusion, serum level of miR-128-2 serves as a noninvasive biomarker for the overall survival of patients with hepatocellular carcinoma.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

Circulating microRNAs as potential biomarkers for smoking-related interstitial fibrosis

Numerous efforts have been made to indentify reliable and predictive biomarkers to detect the early signs of smoking-induced lung disease. Using 6-month cigarette smoking in mice, we have established smoking-related interstitial fibrosis (SRIF). Microarray analyses and cytokine/chemokine biomarker measurements were made to select circulating microRNAs (miRNAs) biomarkers. We have demonstrated that specific miRNAs species (miR-125b-5p, miR-128, miR-30e, and miR-20b) were significantly changed, both in the lung tissue and in plasma, and exhibited mainstream (MS) exposure duration-dependent pathological changes in the lung. These findings suggested a potential use of specific circulating miRNAs as sensitive and informative biomarkers for smoking-induced lung disease.     

Circulating microRNAs as Specific Biomarkers for Breast Cancer Detection

Background

We previously showed microRNAs (miRNAs) in plasma are potential biomarkers for colorectal cancer detection. Here, we aimed to develop specific blood-based miRNA assay for breast cancer detection.

Methodology/Principal Findings

TaqMan-based miRNA profiling was performed in tumor, adjacent non-tumor, corresponding plasma from breast cancer patients, and plasma from matched healthy controls. All putative markers identified were verified in a training set of breast cancer patients. Selected markers were validated in a case-control cohort of 170 breast cancer patients, 100 controls, and 95 other types of cancers and then blindly validated in an independent set of 70 breast cancer patients and 50 healthy controls. Profiling results showed 8 miRNAs were concordantly up-regulated and 1 miRNA was concordantly down-regulated in both plasma and tumor tissue of breast cancer patients. Of the 8 up-regulated miRNAs, only 3 were significantly elevated (p<0.0001) before surgery and reduced after surgery in the training set. Results from the validation cohort showed that a combination of miR-145 and miR-451 was the best biomarker (p<0.0001) in discriminating breast cancer from healthy controls and all other types of cancers. In the blind validation, these plasma markers yielded Receiver Operating Characteristic (ROC) curve area of 0.931. The positive predictive value was 88% and the negative predictive value was 92%. Altered levels of these miRNAs in plasma have been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 96%.

Conclusions

These results suggested that these circulating miRNAs could be a potential specific biomarker for breast cancer screening.     

A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer

Background

To date, there are no highly sensitive and specific minimally invasive biomarkers for detection of breast cancer at an early stage. The occurrence of circulating microRNAs (miRNAs) in blood components (including serum and plasma) has been repeatedly observed in cancer patients as well as healthy controls. Because of the significance of miRNA in carcinogenesis, circulating miRNAs in blood may be unique biomarkers for early and minimally invasive diagnosis of human cancers. The objective of this pilot study was to discover a panel of circulating miRNAs as potential novel breast cancer biomarkers.

Methodology/Principal Findings

Using microarray-based expression profiling followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 women with early stage breast cancer (10 Caucasian American (CA) and 10 African American (AA)) and 20 matched healthy controls (10 CAs and 10 AAs). Using the significance level of p<0.05 constrained by at least two-fold expression change as selection criteria, we found that 31 miRNAs were differentially expressed in CA study subjects (17 up and 14 down) and 18 miRNAs were differentially expressed in AA study subjects (9 up and 9 down). Interestingly, only 2 differentially expressed miRNAs overlapped between CA and AA study subjects. Using receiver operational curve (ROC) analysis, we show that not only up-regulated but also down-regulated miRNAs can discriminate patients with breast cancer from healthy controls with reasonable sensitivity and specificity. To further explore the potential roles of these circulating miRNAs in breast carcinogenesis, we applied pathway-based bioinformatics exploratory analysis and predicted a number of significantly enriched pathways which are predicted to be regulated by these circulating miRNAs, most of which are involved in critical cell functions, cancer development and progression.

Conclusions

Our observations from this pilot study suggest that the altered levels of circulating miRNAs might have great potential to serve as novel, noninvasive biomarkers for early detection of breast cancer.     

Elevated circulating microRNA-210 levels in patients with hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations: a potential new biomarker

Pulmonary arteriovenous malformations (PAVMs), which can lead to life-threatening bleeding and other complications, have been reported to occur in 30–50% of patients with hereditary hemorrhagic telangiectasia (HHT). Circulating microRNAs (miRNAs) have emerged as new biomarkers for human diseases. This study was conducted to explore circulating miRNAs as biomarkers for the screening of HHT patients with PAVMs. MicroRNA array analysis revealed eight altered circulating miRNAs in patients with PAVMs. Real time RT-PCR showed that the levels of circulating miR-210 were significantly elevated in HHT patients with PAVMs but not changed in patients without PAVMs as compared with healthy controls. Circulating miR-210 therefore may be used as a new and sensitive biomarker for the screening of patients with HHT for clinically significant PAVMs.     

Comprehensive proteome analysis of malignant pleural effusion for lung cancer biomarker discovery by using multidimensional protein identification technology

Malignant pleural effusion (MPE) obtained from lung adenocarcinoma may contain potentially useful biomarkers for detection of lung cancer. In this study, we used a removal system for high-abundance proteins followed by one-dimensional SDS-PAGE combined with nano-LC-MS/MS to generate a comprehensive MPE proteome data set with 482 nonredundant proteins. Next, we integrated the MPE proteome and secretome data sets from three adenocarcinoma cell lines, with a view to identifying potential PE biomarkers originating from malignant cells. Four potential candidates, alpha-2-HS-glycoprotein (AHSG), angiogenin, cystatin-C, and insulin-like growth factor-binding protein 2, (IGFBP2), were isolated for preclinical validation using ELISA. Both AHSG and IGFBP2 levels were increased in lung patients with MPE (n = 68), compared to those with nonmalignant pleural effusion (n = 119). Notably, the IGFBP2 level was higher in MPE, compared with that in benign diseases (bacteria pneumonia and tuberculosis pleuritis), and significantly associated with malignancy, regardless of the cancer type. Our data additionally support an extracellular function of IGFBP2 in migration in lung cancer cells. These findings collectively suggest that the adenocarcinoma MPE proteome provides a useful data set for malignancy biomarker research.     

Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS-PAGE combined with MALDI-TOF MS. Among the 325 proteins identified, collapsin response mediator protein-2 (CRMP-2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP-2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP-2-positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP-2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 +/- 34.6 vs. 40.2 +/- 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP-2 may be a novel CRC biomarker.     

A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer

Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.     

Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.     

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep–1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX–2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC–70, protein disulfide isomerase (PDI), and heat shock protein–27 (HSP27), were exclusively up-regulated in SK-Hep–1 cells. Elevated levels of ANX–4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep–1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.     

A new versatile peptide-based size exclusion chromatography platform for global profiling and quantitation of candidate biomarkers in hepatocellular carcinoma specimens

Disease biomarkers are predicted to be in low abundance; thus, the most crucial step of biomarker discovery is the efficient fractionation of clinical samples into protein sets that define disease stages and/or predict disease development. For this purpose, we developed a new platform that uses peptide-based size exclusion chromatography (pep-SEC) to quantify disease biomarker candidates. This new platform has many advantages over previously described biomarker profiling platforms, including short run time, high resolution, and good reproducibility, which make it suitable for large-scale analysis. We combined this platform with isotope labeling and label-free methods to identify and quantitate differentially expressed proteins in hepatocellular carcinoma (HCC) tissues. When we combined pep-SEC with a gas phase fractionation method, which broadens precursor ion selection, the protein coverage was significantly increased, which is critical for the global profiling of HCC specimens. Furthermore, pep-SEC-LC-MS/MS analysis enhanced the detection of low-abundance proteins (e.g. insulin receptor substrate 2 and carboxylesterase 1) and glycopeptides in HCC plasma. Thus, our pep-SEC platform is an efficient and versatile pre-fractionation system for the large-scale profiling and quantitation of candidate biomarkers in complex disease proteomes.