Identification of hydrophobic proteins as biomarker candidates for colorectal cancer

Nowadays, colorectal cancer is one of the major causes of cancer death in Western countries. Due to the lack of biomarkers with clinical utility for this pathology, and considering that membrane and hydrophobic proteins have not been studied in depth, we performed a prefractionation of colorectal tissues prior to two-dimensional gel electrophoresis in order to identify hydrophobic proteins differentially expressed in colorectal cancer patients. Fractions enriched in hydrophobic proteins were obtained from healthy mucosa and tumor tissue by a specific extraction method based on temperature-dependent phase partitioning with Triton X-114. Proteins were separated by two-dimensional gel electrophoresis and gels were silver-stained, scanned and compared using the PDQuest software. Those spots presenting significantly different abundance were submitted to mass spectrometry for protein identification. Alterations in the expression of cytoskeletal proteins, including a decrease of vimentin and the absence of desmin, were found. We also detected alterations in antioxidant and transport proteins, chaperones, and in two isoforms of the calcium-binding protein S100A6. On the other hand, vimentin was chosen to corroborate the electrophoretic results by specific immunodetection. Most of the altered proteins have been related to cellular membranes, many of them to lipid rafts microdomains in the plasma membrane, and they have also been implicated in the control of cell proliferation, apoptosis, or metastasis. In conclusion, all the proteins found altered in colorectal tumor samples could be considered as candidates for future studies focused on their utility as markers for colorectal diagnosis and prognosis, or as targets for colorectal cancer therapy.     

Identification of alphaB-crystallin, a biomarker of renal cell carcinoma by SELDI-TOF MS

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.     

Quantitative proteomics analysis reveals that proteins differentially expressed in chronic pancreatitis are also frequently involved in pancreatic cancer

The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.     

Global expression study in colorectal cancer on proteins with alkaline isoelectric point by two-dimensional difference gel electrophoresis

Colorectal cancer is one of the leading causes of cancer death worldwide. To identify candidates for biomarkers and therapeutic targets, we investigated the proteome of colorectal cancer tissues. Using 2D-DIGE in combination with our original large format electrophoresis apparatus, we compared surgically resected normal and tumor tissues from 53 patients with colorectal cancer. We focused on proteins with an alkaline pI using IPG gels for the alkaline range. We observed 1687 protein spots, and found 100 spots with statistical (p<0.01) and significant (>2-fold) differences between the normal and the tumor tissue groups. Among these 100 protein spots, five showed a different intensity between tumor tissues from the stage-II and the stage-III patients. MS experiments revealed that these 100 protein spots corresponded to 58 unique proteins. These included six proteins which had not been previously reported to be associated with colorectal cancer. Among these proteins, five were not reported in any type of malignancy. IEF/western blotting confirmed the differences in protein expression between the normal and the tumor tissues. These results may provide an insight for biomarker development and drug target discovery in colorectal cancer.     

The analysis of microRNA-34 family expression in human cancer studies comparing cancer tissues with corresponding pericarcinous tissues

Recently many studies have focused on the microRNA-34 (miR-34) family expression in various cancers; nevertheless, the controversial results of these studies still exist in identifying miR-34 members as new biomarkers of cancers. Therefore, we carried out this comprehensive meta-analysis of published studies that compared the miR-34 family expression profiles between cancer tissues and paired neighboring noncancerous tissues to systemically evaluate the findings globally and address the inconsistencies of pertinent literatures. The data included in this article were collected from Embase, PubMed and Web of Science up to December 2013. To overcome the difficulties that many raw data were unavailable and study methods were different, a vote-counting strategy was adopted to identify consistent markers in our analysis. Ultimately, a total of 23 cancers were reported in the 61 eligible studies, of which 46 studies provided fold-change value information. In the consistently reported cancer types, non-small cell lung cancer (NSCLC), glioma and nasopharyngeal carcinoma (NPC) ranked at the top with down-regulated feature. Cervical neoplasm was consistently reported to be over-expressed in the panel of each member of miR-34s. Subgroup analysis of miR-34 family expression demonstrated that colorectal cancer (CRC), gastric cancer (GC), hepatocellular carcinoma (HCC) and prostate cancer (PCa) were most frequently reported with inconsistent regulations. Our meta-analysis showed that miR-34 family members could be expected to become potential diagnostic and prognostic biomarkers in some types of human cancers. Further well-designed and larger sample studies are surely warranted to identify the role of the miR-34 family in the occurrence and development of tumors.     

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.     

Differential expression of mimecan and thioredoxin domain-containing protein 5 in colorectal adenoma and cancer: a proteomic study

Adenoma is the major precursor lesion of colorectal cancer, one of the most common cancers worldwide. The elucidation of the molecular mechanism underlying adenoma is essential for early detection, prevention, and intervention of colorectal cancer. Using a combination of two-dimensional gel electrophoresis and mass spectrometry, we identified 27 differentially expressed proteins in adenoma, compared with matched normal mucosa and cancer tissue. Seventeen proteins were upregulated and six downregulated in adenoma when compared with the same proteins in individual-matched normal mucosa. Four were downregulated, but none upregulated in adenoma when compared with the same proteins in matched cancer tissue. Two novel proteins, mimecan and thioredoxin domain-containing protein 5 (TXNDC5), were further validated by Western blot in 8 colorectal adenomas and 19 cancers that were matched with normal mucosa. All adenoma and cancer tissues did not express mimecan, but all normal mucosa did (P < 0.01). In contrast, TXNDC5 was significantly upregulated in colorectal adenoma and cancer tissues as compared with that in normal mucosa (P < 0.05). This study clearly demonstrated that absence of mimecan and upregulation of TXNDC5 are involved in the early development of colorectal cancer. Thus, the differentially expressed proteins might serve as potential biomarkers for colorectal cancer detection and intervention.     

Identification of HSP27 as a potential tumor marker for colorectal cancer by the two-dimensional polyacrylamide gel electrophoresis

The identification of specific biomarkers for colorectal cancer would provide the basis for early diagnosis, prognosis, therapy, as well as clues for understanding the molecular mechanisms governing cancer progression. This study was designed to use comparative proteomics technology to find the differentially expressed proteins between human colorectal carcinoma and the corresponding normal tumor-adjacent colorectal tissues. We have used the highly sensitive two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF–MS) for the identification of proteins differentially expressed in tumoral and neighboring normal mucosa. We have detected differences in abundance of 42 proteins with statistical variance of the tumor versus normal spot volume ratio within the 95th confidence level (Student’s t-test; P < 0.05). 10 out of 42 analyzed proteins were unambiguously identified by MS coupled with database interrogation as being differentially expressed in colorectal cancer. Of the 10 newly implicated proteins, HSP27 was chosen for detailed analysis. Preliminary studies demonstrated that the differentially expressed proteins found by 2-DE could be confirmed and validated by western blotting and immunohistochemistry analyses in those few cases. The results suggest that HSP27 might be a potential biomarker for early diagnosis, prognosis, monitoring in the therapy of colorectal carcinoma.     

Potential protein markers for nutritional health effects on colorectal cancer in the mouse as revealed by proteomics analysis

It is suggested that colorectal cancer might be prevented by changes in diet, and vegetable consumption has been demonstrated to have a protective effect. Until now, little is known about the effects of vegetable consumption at the proteome level. Therefore, the effect of increased vegetable intake on the protein expression in the colonic mucosa of healthy mice was studied. Aim was to identify the proteins that are differentially expressed by increased vegetable consumption and to discriminate their possible role in the protection against colorectal cancer. Mice were fed four different vegetable diets, which was followed by analysis of total cellular protein from colonic mucosal cells by a combination of 2-DE and MS. We found 30 proteins that were differentially expressed in one or more diets as compared to the control diet. Six could be identified by MALDI-TOF MS: myosin regulatory light chain 2, carbonic anhydrase I, high-mobility group protein 1, pancreatitis-associated protein 3, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase oligomycin sensitivity conferral protein. Alterations in the levels of these proteins agree with a role in the protection against colon cancer. We conclude that these proteins are suitable markers for the health effect of food on cancer. The observed altered protein levels therefore provide support for the protective effects of vegetables against colorectal cancer.     

Identification of biomarkers for colorectal cancer through proteomics-based approaches

The early detection of colorectal cancer is one of the great challenges in the battle against this disease. However, owing to its heterogeneous character, single markers are not likely to provide sufficient diagnostic power to be used in colorectal cancer population screens. This review provides an overview of recent studies aimed at the discovery of new diagnostic protein markers through proteomics-based approaches. It indicates that studies that start with the proteomic analysis of tumor tissue or tumor cell lines (near the source) have a high potential to yield novel and colorectal cancer-specific biomarkers. In the next step, the diagnostic accuracy of these candidate markers can be assessed by a targeted ELISA assay using serum from colorectal cancer patients and healthy controls. Instead, direct proteomic analysis of serum yields predominantly secondary markers composed of fragments of abundant serum proteins that may be associated with tumor-associated protease activity, and alternatively, immunoproteomic analysis of the serum antibody repertoire provides a valuable tool to identify the molecular imprint of colorectal cancer-associated antigens directly from patient serum samples. The latter approach also allows a relatively easy translation into targeted assays. Eventually, multimarker assays should be developed to reach a diagnostic accuracy that meets the stringent criteria for colorectal cancer screening at the population level.     

Meta-analysis of microRNA-183 family expression in human cancer studies comparing cancer tissues with noncancerous tissues

MicroRNA-183 (miR-183) family is proposed as promising biomarkers for early cancer detection and accurate prognosis as well as targets for more efficient treatment. The results of their expression feature in cancer tissues are inconsistent and controversy still exists in identifying them as new biomarkers of cancers. Therefore, to systemically evaluate the most frequently reported cancers in which miR-183 family members were up- or down-regulated is critical for further investigation on physiological impact of its aberrant regulation in specific cancers. The published studies that compared the level of miR-183 family expression in cancer tissues with those in noncancerous tissues were reviewed by the meta-analysis with a vote-counting strategy. Among the 49 included studies, a total of 18 cancers were reported, with 11 cancers reported in at least two studies. In the panel of miR-183 family members' expression analysis, colorectal cancer and prostate cancer ranked at the top among consistently reported cancer types with up-regulated feature. Bladder cancer, lung cancer and hepatocellular carcinoma were the third most frequently reported cancer types with significant over-expression of miR-96, miR-182 and miR-183 respectively. Breast cancer and gastric cancer were presented with inconsistent regulations and the members of this family had their own distinct regulated features in other different cancers. MiR-183 family, either individually or as a cluster, may be useful prognostic markers and/or therapeutic targets in several cancers. Further studies and repeat efforts are still required to determine the role of miR-183 family in various cancer progressions.     

Expression profiling of more than 3500 proteins of MSS-type colorectal cancer by stable isotope labeling and mass spectrometry

An efficient means of identifying protein biomarkers is essential to proper cancer management. A well-characterized proteome resource holds special promise for the discovery of novel biomarkers. However, quantification of the differences between physiological conditions together with deep down profiling has become increasingly challenging in proteomics. Here, we perform expression profiling of the colorectal cancer (CRC) proteome by stable isotope labeling and mass spectrometry. Quantitative analysis included performing mTRAQ and cICAT labeling in a pooled sample of three microsatellite stable (MSS) type CRC tissues and a pooled sample of their matched normal tissues. We identified and quantified a total of 3688 proteins. Among them, 1487 proteins were expressed differentially between normal and cancer tissues by higher than 2-fold; 1009 proteins showed increased expression in cancer tissue, whereas 478 proteins showed decreased expression. Bioinformatic analysis revealed that our data were largely consistent with known CRC relevant signaling pathways, such as the Wnt/β-catenin, caveolar-mediated endocytosis, and RAN signaling pathways. Mitochondrial dysfunction, known as the Waburg hypothesis, was also confirmed. Therefore, our data showing alterations in the proteomic profile of CRC constitutes a useful resource that may provide insights into tumor progression with later goal of identifying biologically and clinically relevant marker proteins. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Proteomic analysis of conditioned media from the PC3, LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate prostate cancer biomarkers

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.     

Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin beta1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.