Discovery of Tumor Markers for Gastric Cancer by Proteomics

Gastric cancer (GC) has a high rate of morbidity and mortality among various cancers worldwide. The development of noninvasive diagnostic methods or technologies for tracking the occurrence of GC is urgent, and searching reliable biomarkers is considered.This study intended to directly discover differential biomarkers from GC tissues by two-dimension-differential gel electrophoresis (2D-DIGE), and further validate protein expression by western blotting (WB) and immunohistochemistry (IHC).Pairs of GC tissues (gastric cancer tissues and the adjacent normal tissues) obtained from surgery was investigated for 2D-DIEG.Five proteins wereconfirmed by WB and IHC, including glucose-regulated protein 78 (GRP78), glutathione s-transferase pi (GSTpi), apolipoprotein AI (ApoAI), alpha-1 antitrypsin (A1AT) and gastrokine-1 (GKN-1). Among the results, GRP78, GSTpi and A1ATwere significantlyup-regulated and down-regulated respectively in gastric cancer patients. Moreover, GRP78 and ApoAI were correlated with A1AT for protein expressions.This study presumes these proteins could be candidates of reliable biomarkers for gastric cancer.     

Proteomic analysis of cancer tissues: shedding light on carcinogenesis and possible biomarkers

Lung, gastric, colorectal, pancreatic, and esophageal cancers, as well as hepatocellular carcinoma (HCC), were the six most common and highly fatal cancers for Japanese men in Japan in 2003, while for women uterine cervical cancer could also be added to this list. To identify diagnostic or therapeutic biomarkers for these cancers, investigators are nowadays performing proteomic analyses of cancer tissues and cells, and revealing a large number of molecules which are diagnostic, prognostic and informative of carcinogenesis. From reports of proteomic analyses of cancerous tissues and noncancerous tissues sampled from HCC, and pancreatic, esophageal, gastric, colorectal, lung and uterine cervical cancers, we classified the proteins into digestive enzymes, growth factors, cell adhesion molecules, calcium-binding proteins, proteases, protease inhibitors, transporter proteins, structural molecules, apoptosis inhibitor, molecular chaperone, as well as proteins related to cell growth, cell differentiation, cell transformation, tumor invasion, carcinogen metabolism, and others. The aim of this study was to understand carcinogenesis of major cancers from a proteomics perspective using samples from cancer patients, and to elucidate their tumor biomarkers.     

Comprehensive proteomic analysis of human pancreatic juice

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.     

Telomerase activity could be used as a marker for neoplastic transformation in gastric adenocarcinoma: but it does not have a prognostic significance

Telomerase activity is responsible for telomere maintenance and is believed to be crucial in most immortal cells and cancer cells; however, its clinicopathological significance in gastric cancer remains to be clarified. The aim of the present study was to assess whether malignant progression of gastric adenocarcinoma correlates with telomerase activity. We also investigated the correlation between telomerase activity and histopathological findings. We examined telomerase activity in tumor specimens and adjacent normal tissues from 43 patients with gastric adenocarcinoma. Telomerase activity was measured quantitatively by the TRAPEZE Gel Based Telomerase Detection Kit. Approximately 98% of the tumor tissues were telomerase positive, but telomerase activity was detected not only in tumor tissues but also in normal gastric mucosa. Although telomerase activity was found to be higher in tumor samples than normal tissue for each subject, we could not find a general cut-off level for telomerase activity in gastric adenocarcinoma. In addition, telomerase activity was not correlated with tumor invasion, lymph node involvement and histological stage. Our results support the idea that telomerase reactivation is a common event in gastric adenocarcinoma and it is not related to histopathological parameters. Since it is difficult to set a cut-off level for this type of cancer, we suggest that the prognostic utility of telomerase assay has not yet reached the clinic in terms of predicting outcome for patients with gastric adenocarcinoma. For the assessment of gastric carcinoma, telomerase activity should be evaluated in both tumor and normal tissues, because normal gastric mucosa samples show appreciable telomerase activity.     

A proteomics analysis of cell signaling alterations in colorectal cancer

To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.     

Two‐dimensional gel electrophoresis of gastric tissue in an alkaline pH range

2DE in combination with MS has facilitated the discovery of several proteins with altered abundance in gastric cancer. While acidic and wide pH ranges have been widely investigated, analysis in the alkaline pH range has not been specifically performed in gastric cancer to date. In the present study, we initially optimized the 2DE in alkaline pH range (pH 7–11) for gastric tissue samples. Using a modified lysis buffer, we analyzed pooled nontumor and tumor samples for proteins with altered abundance in gastric adenocarcinoma. We successfully identified 38 silver‐stained spots as 24 different proteins. Four of these were chosen for investigation with immunoblotting on individual paired samples to determine whether the changes seen in 2DE represent the overall abundance of the protein or possibly only a single form. While mitochondrial trifunctional protein (MTP) subunits were decreased in 2DE gels, immunoblotting identified their overall abundance as being differently dysregulated: in the gastric tumor samples, the MTP‐α subunit was decreased, and the MTP‐β subunit was increased. On the other hand, heterogenous nuclear ribonucleoprotein M and galectin‐4 were increased in the gastric tumor samples in both 2DE and immunoblotting.     

Identification of human hepatocellular carcinoma-related biomarkers by two-dimensional difference gel electrophoresis and mass spectrometry

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death throughout the world. Although hepatitis B or C viral infections are main risk factors for HCC, the molecular mechanisms leading to HCC formation have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. In this study, we employed two-dimensional difference gel electrophoresis (2D-DIGE) combined with nano flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) to investigate differentially expressed proteins in HCC. For each of eight HCC patients, Cy3-labeled proteins isolated from tumor tissue were combined with Cy5-labeled proteins isolated from the surrounding nontumor tissue and separated by 2D gel electrophoresis along with a Cy2-labeled mixture of all tumor and nontumor samples as an internal standard. Thirty-four protein spots corresponding to 30 different proteins were identified by nanoLC-MS/MS as showing significant change (paired t-test, p < 0.05) in the level of expression between tumor and nontumor tissues. Sixteen proteins were up-regulated and 14 were down-regulated in HCC; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport and trafficking, amino acid metabolism, iron and xenobiotic metabolism, ethanol metabolism, cell cycle regulation, cytoskeleton, and stress. A remarkable finding is the up-regulation of 14-3-3gamma protein in HCC. 14-3-3 isoforms had been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. In conclusion, 2D-DIGE is an efficient strategy that enables us to identify differentially expressed proteins in HCC. Identification of potential biomarkers, such as the pinpointing of 14-3-3gamma in our findings, may provide further useful insights into the pathogenesis of HCC.     

Sequential alterations in gastric biopsies and tumor tissues support the multistep process of carcinogenesis

Gastric cancer is the second leading cause of cancer related death worldwide. In the UAE, recent data show an increase in the number of patients with gastric cancer highlighting the need for greater understanding of its pathogenesis. Gastric cancer is generally believed to develop on a background of chronic atrophic gastritis which eventually leads to intestinal metaplasia, dysplasia and finally invasive carcinoma. Recently this multistep process of carcinogenesis has been challenged. Therefore, the aim of this study is to define alterations in antral mucosal biopsies and cancer tissues to investigate whether they could be used to assemble a tissue array supporting the multistep model of carcinogenesis. Gastric mucosal tissues were obtained from informed individuals undergoing endoscopy (for upper gastrointestinal symptoms) and gastrectomy (for adenocarcinoma) in Tawam Hospital. All tissues were processed for microscopic examination. Eighty nine antral biopsies were categorized as: normal (33%), mild superficial gastritis (34%) and severe atrophic gastritis (33%). About 5% of the latter exhibited evidence of intestinal metaplasia. Cancer tissues obtained from three patients were microscopically examined in three regions: safe resected margin, tumor edge and tumor center. Progressive changes in mucosal thickness, dysplasia and cellular transformation were observed, and when compared with alterations in biopsies, all appeared to represent a continuum of progression toward invasive adenocarcinoma. In conclusion, the tissue array presented in this study supports the multistep process of gastric carcinogenesis and will be helpful in examining the expression pattern of tumor markers or molecules that could help in the early detection of gastric cancer.     

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 tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.     

Molecular characteristics and chromatin texture features in acute promyelocytic leukemia

Background

Ezrin is a cytoskeletal protein that is involved in tumor growth and invasion. It has been suggested that Ezrin expression plays an important role in tumor metastasis. This study is aimed to investigate the clinicopathological significance of Ezrin overexpression in gastric adenocarcinomas.

Methods

Ezrin protein expression was examined by immunohistochemistry in 26 normal gastric mucosa, 32 dysplasia, and 277 gastric adenocarcinomas. The relationship between Ezrin expression and the clinicopathological features of gastric cancers was analyzed. In addition, a gastric cancer cell line, MKN-1, was also used for immunofluorescence staining to evaluate the distribution of Ezrin protein.

Results

Ezrin protein located in the cytoplasm and/or membrane in the migrating gastric cancer cells, and it mainly concentrated at the protrusion site; however, only cytoplasmic distribution was observed in the non-migrating cancer cells by immunofluorescence staining. The positive rate of Ezrin protein expression was significantly higher in gastric adenocarcinoma and dysplasia compared with that in the normal gastric mucosa. Moreover, expression frequency of Ezrin protein increased significantly in lymph node metastasis and late clinical stages. Consistently, strong expression of Ezrin was significantly correlated with poor prognosis of gastric cancer.

Conclusion

The detection of Ezrin expression can be used as the marker for early diagnosis and prognosis of gastric adenocarcinoma.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2303598677653946     

Comparative analysis of the protein profiles from primary gastric tumors and their adjacent regions: MAWBP could be a new protein candidate involved in gastric cancer

The study of tumor biomarkers is generally facilitated by the adoption of proteomic strategies. With limitations of techniques and individual varieties of biological samples, the biomarkers for gastric cancer (GC) have not reached a common agreement derived from the proteomic investigations. Herein, we reported a new set of data for screening the biomarkers from the gastric tissues, on the basis of the proteomic strategy developed in our laboratory. Ten pairs of the clinic samples were collected and treated with protein extraction. The gastric proteins were well-resolved by 2-DE, and the GC-associated proteins were identified by MALDI-TOF/TOF MS following image analysis, including 12 up-regulated and 13 down-regulated unique ones. MAWBP was found to be one of the new GC proteins which appeared with lower expression in the GC tissues. We expanded a systematical examination to deeply pursue the relevance between MAWBP and GC. Quantitatively, we measured the expression of MAWBP with Western blot and Real-Time PCR. Extendedly, we estimated the existence of MAWBP with immunohistochemical staining in a large number of the GC cases. Specifically, we inquired whether MAWD, a protein with high affinity to MAWBP, could coexpress and interact with MAWBP in vivo. On the basis of all the results, we concluded that MAWBP could be a new GC-related protein even though its physiological roles remain unexplored.     

Claudins upregulation in human colorectal cancer

In colorectal cancer tight junction molecular and morphological alterations are poorly understood. In this study, adenocarcinoma tissues and their paired normal mucosa (n = 12) were analyzed for tight junction alterations molecular. The expression of claudin-1, -3 and -4 was upregulated 5.7-, 1.5- and 2.4-fold, respectively, in colorectal tumor tissues in comparison to the normal ones. Although tight junction remains in the cancerous epithelium, its barrier function was altered. Despite claudins overexpression, paracellular permeability to ruthenium red was increased and a significant disorganization of tight junction strands was observed in freeze fracture replicas. Whereas the functional significance of claudin overexpression in colorectal cancer is unclear, these proteins can become potential markers and targets in colorectal cancer.     

Genetic and epigenetic alterations of tumor suppressor and tumor-related genes in gastric cancer

Both genetic and epigenetic alterations of tumor suppressor and tumor-related genes involved in the pathogenesis of gastric cancer are reviewed here, and molecular pathways of gastric carcinogenesis are proposed. Gastric carcinomas are believed to evolve from native gastric mucosa or intestinal metaplastic mucosa that undergoes genetic and epigenetic alterations involving either the suppressor pathway (defects in tumor suppressor genes) or mutator pathway (defects in DNA mismatch repair genes). Methylation of E-cadherin in native gastric mucosa results in undifferentiated carcinomas (suppressor pathway), while methylation of hMLHI results in differentiated foveolar-type carcinomas (mutator pathway). The majority of differentiated gastric carcinomas however, arise from intestinal metaplastic mucosa and exhibit structural alterations of tumor suppressor genes, especially p53. They appear to be related to chronic injury, perhaps due to Helicobacter pylori infection. Approximately 20% of differentiated carcinomas (ordinary-type) have evidence of mutator pathway tumorigenesis. Mutations of E-cadherin are mainly involved in the progression of differentiated carcinomas to undifferentiated tumors. The molecular pathways of gastric carcinogenesis depend on the histological background, and gastric carcinomas show distinct biological behaviors as a result of discernible cellular genetic and epigenetic alterations.     

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical-pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.     

淋巴道转移相关标志物与癌

肿瘤早期的淋巴道转移是恶性肿瘤患者高死亡率的主要原因,其机制复杂,一直是肿瘤研究的难点. 肿瘤淋巴道转移特异性蛋白标记物的发现有助于揭示恶性肿瘤早期发病机制、早期临床诊断和治疗. 本文综述了近年来肝癌、胃癌、食管癌、结肠癌、乳腺癌、肺癌、鼻咽癌和前列腺癌淋巴道转移研究所取得的蛋白质组学成果,并对发现的与以上几种肿瘤淋巴道转移相关的蛋白标记物及其在肿瘤的发生、浸润、转移及病人预后中作用及机制进行了探讨.     

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.