OCT-1 overexpression is associated with poor prognosis in patients with well-differentiated gastric cancer.

Octamer transcription factor-1 (OCT-1) is a well-known transcription factor that is reportedly overexpressed in intestinal metaplasia and gastric carcinoma in the intestine. In this study, we investigated OCT-1 overexpression as a prognostic factor for gastric cancer. The association between OCT-1 overexpression (detected using immunohistochemistry) and clinicopathological features including survival was evaluated. In vitro gain-of-function approaches were utilized to assess the function of OCT-1 in malignancy. Analysis of OCT-1 expression in patients with gastric cancer with well-differentiated carcinoma as per the World Health Organization classification showed that OCT-1 overexpression was correlated with advanced tumor invasion (58.8 % of patients with advanced tumor invasion vs. 21.2 % of patients with early tumor invasion; p<0.01), lymph node metastasis (63.9 % of patients with metastasis vs. 24.1 % of those without; p=0.015), and cancer recurrence (83.3 % of patients with recurrence vs. 25.4 % of those without; p<0.01), as well as a lower survival rate (62.8 vs. 87.9 Mo; p<0.01). However, there were no significant differences in the levels of OCT-1 expression in gastric cancer patients with other carcinoma types (p>0.05). Furthermore, we found that the proliferation rate of OCT-1-overexpressing MKN-45 cells was higher than that of the control cells. OCT-1 overexpression may be a marker for poor prognosis in patients with well-differentiated gastric adenocarcinoma.

Pyrophosphatase overexpression is associated with cell migration, invasion, and poor prognosis in gastric cancer.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate to form orthophosphate. Pyrophosphate can substitute for ATP under certain circumstances. We previously conducted a proteomic analysis to investigate tumor-specific protein expression in gastric cancer, and PPase was identified as a potential gastric tumor-specific marker; it was therefore selected for further study. Clinicopathological analysis, using proteomic analysis and immunohistochemistry, was used to validate PPase as a prognostic marker in gastric cancers. Proteomic analysis showed that PPase was overexpressed in patients with lymph node (LN) metastases and high tumor node metastasis (TNM) stages (p < 0.05). Based on immunohistochemistry, patients whose tumors overexpressed PPase had higher T stages, LN metastasis, a higher TNM stage, a higher cancer recurrence rate, and shorter survival times than patients whose tumors exhibited PPase underexpression (p < 0.05). Gain-of-function and loss-of-function approaches were employed to examine the malignant phenotypes of PPase-overexpressing or PPase-depleted cells. A decrease in PPase expression caused a significant decrease in gastric cancer cell migration and invasion in vitro, whereas forced overexpression of PPase enhanced migration but not invasion. Our findings indicate that PPase is involved in gastric tumor progression and that PPase may be a useful marker for poor prognosis of human gastric cancers.

Downregulation of CHIP promotes ovarian cancer metastasis by inducing Snail-mediated epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) plays a pivotal role in the conversion of early-stage tumors into invasive malignancies. The transcription factor Snail, an extremely unstable protein whose subcellular levels are regulated by many E3 ubiquitin ligases, promotes EMT as well as associated pathological characteristics including migration, invasion, and metastasis. Through yeast two-hybrid screening, we identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as a novel Snail ubiquitin ligase that interacts with Snail to induce ubiquitin-mediated proteasomal degradation. Inhibition of CHIP expression increases Snail protein levels, induces EMT, and enhances in vitro migration and invasion as well as in vivo metastasis of ovarian cancer cells. In turn, Snail depletion abrogates all phenomena induced by CHIP depletion. Finally, Snail and CHIP expression is inversely correlated in ovarian tumor tissues. These findings establish the CHIP-Snail axis as a post-translational mechanism of EMT and cancer metastasis regulation.

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification.

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P < 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.

Transcriptional abnormality of the hsMAD2 mitotic checkpoint gene is a potential link to hepatocellular carcinogenesis.

MAD2 is localized to kinetochores of unaligned chromosomes, where it inactivates the anaphase-promoting complex/cyclosome, thus contributing to the production of a diffusible anaphase inhibitory signal. Disruption of MAD2 expression leads to defects in the mitotic checkpoint, chromosome missegregation, and tumorigenesis. However, the mechanism by which deregulation and/or abnormality of hsMAD2 expression remains to be elucidated. Here, we clone and analyze a approximately 0.5 kb fragment upstream of hsMAD2 and show that this fragment acts as a strong promoter. Transcriptional dysfunction of hsMAD2 is frequently observed in hepatocellular carcinoma cells, and down-regulation of hsMAD2 protein expression is correlated with transcriptional silencing of the hsMAD2 promoter by hypermethylation. These results imply a relationship between transcriptional abnormality of this mitotic checkpoint gene and mitotic abnormality in human cancers.

Prognostic value of CAPZA1 overexpression in gastric cancer.

F-actin capping protein α1 subunit (CAPZA1) was previously identified in a proteomic analysis of human gastric cancer clinical specimens and selected for further study. The association between CAPZA1 overexpression, detected by immunohistochemistry, and clinicopathological features including survival were evaluated. In vitro gain-of-function and loss-of-function approaches were utilized to assess the function of CPAZA1 in malignancy. Univariate analysis revealed that poorly differentiated disease, according to the World Health Organization (WHO) classification, advanced T stage, positive lymph nodes, high TNM stage, D2 lymph node dissection, adjuvant chemotherapy and CAPZA1 underexpression were significantly associated with cancer-related death (p<0.05); however, only high TNM stage remained significantly associated by multivariate analysis (p<0.01). CAPZA1 overexpression was associated with well differentiated histology, smaller tumor size, lower T stage, absence of lymph node metastasis, lower TNM stage, lower recurrence rate and longer survival time, compared to CAPZA1 underexpression. In vitro, forced expression of CAPZA1 caused a significant decrease in gastric cancer cell migration and invasion, whereas CAPZA1 depletion had the opposite effect. The present study suggests that CAPZA1 could be a marker of good prognosis in gastric cancer and shows that CAPZA1 is associated with decreased cancer cell migration and invasion.

Fatty acid-binding protein 5 promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a rare primary malignant liver tumor with an extremely poor prognosis. Recently its incidence has increased, however, little attention has been directed to factors related to its molecular carcinogenesis, including oncogenes, tumor suppressor genes and cell cycle-related proteins. ICC is generally characterized by strong proliferation, invasion and early metastasis. These biological behaviors of ICC, with respect to the genetic and molecular aspects, remain to be clarified. In this study, we performed a proteomic analysis to identify the proteomic alterations associated with carcinogenesis of ICC. Protein expression profiles of sixteen cases of ICC were compared with those of adjacent non-involved bile duct tissue. Among the 151 protein spots that showed a statistically significant expression difference (P<0.05), there were 50 spots with significantly increased intensity (3-fold increase) and 17 spots with decreased intensity (3-fold decrease) in cancerous tissues. Of these, increased expression of fatty acid-binding protein 5 (FABP5) was further confirmed by western blot analysis and immunohistochemical analysis. Immunohistochemical analysis of FABP5 expression in tumor specimens obtained from 43 patients with mass-forming (MF) type ICC showed a positive correlation of FABP5 immunoreactivity with tumor size (P=0.047), lymph node metastasis (P=0.013), angioinvasion (P=0.032) and staging (P=0.007). In addition, silencing FABP5 with short hairpin RNA (shRNA) suppressed cell proliferation and invasiveness in HuCCT1 cells, and conversely, overexpression of FABP5 in FABP5-negative Hep3B cells increased cell proliferation and invasiveness. Our study shows that FABP5 is significantly overexpressed in ICC combined lymph node metastasis and is involved in cell proliferation and invasion in vitro. Our data suggest that FABP5 may be associated with tumor progression in ICC.

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer.

PURPOSE: The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. EXPERIMENTAL DESIGN: We performed 2-D gel electrophoresis and MALDI-TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. RESULTS: Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). CONCLUSION AND CLINICAL RELEVANCE: Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.

Galectin-1 expression in cancer-associated stromal cells correlates tumor invasiveness and tumor progression in breast cancer.

Understanding the molecular background of breast cancer biology is critical in developing new biomarkers for earlier diagnosis and more optimized treatment. We performed a proteomic analysis of human breast carcinoma tissues to investigate the tumor-specific protein expression in breast carcinoma. Using 2-dimensional electorphoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), we were able to identify a list of proteins which are upregulated in cancerous tissue. There was significant increase of galectin-1 expression in all cancerous tissues compared to noncancerous tissues, and its increased expression was further confirmed by western blot immunostaining. Subsequent immunohistochemical staining against galectin-1 in 105 breast cancer specimens showed significant correlation between galectin-1 expression in cancer-associated stromal cells and tumor invasiveness, T stage, TNM stage, and axillary lymph node metastasis. Galectin-1 expressionin cancer cells showed no correlation to above-mentioned pathologic variables. Hormonal receptor status and galectin-1 expression showed no correlation. This study demonstrates the upregulation of galectin-1 in breast carcinoma tissues and the clinical significance of galectin-1 in breast cancer patients. Our data supports the recently highlighted roles of galectin-1 in cancer-associated stroma and in tumor immune privilege.