Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients’ survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.     

Low CA II expression is associated with tumor aggressiveness and poor prognosis in gastric cancer patients

Background: Carbonic anhydrase II is present in normal gastric mucosa; thus, this study aimed to investigate whether its expression persisted in neoplastic gastric tissues, as well as its prognostic value for gastric cancer patients. Methods: The protein CA II expression pattern was retrospectively analyzed by immunohistochemistry in 181 gastric cancer patients who had undergone gastrectomy. The relationship between the CA II expression level and clinicopathological parameters was investigated. Survival analysis according to CA II expression was measured by Kaplan-Meier analysis. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic value of CA II expression. Results: CA II expression was significantly decreased in gastric cancer tissues compared with normal stomach mucosa. Low expression was significantly associated with tumor size, depth of invasion, lymph node involvement, distant metastasis and TNM stage, and it predicted poor survival in gastric cancer patients. Moreover, CA II was an independent prognosis indicator for the overall survival of gastric cancer patients. Conclusions: The down-regulation of CA II expression was observed in gastric cancer and may serve as an independent prognostic factor for the overall survival of gastric cancer patients.     

Loss of RKIP expression is associated with poor survival in GISTs

Gastrointestinal stromal tumours (GISTs) are rare mesenchymal tumours of the digestive tract and are commonly driven by oncogenic mutations in KIT and PDGFRA genes. Tumour size, location, mitotic index and KIT/PDGFRA mutations are the most important prognostic parameters in GISTs. However, additional studies screening for new molecular prognostic markers in GISTs are missing. Raf kinase inhibitor protein (RKIP) has been considered as a suppressor of metastasis and a prognostic marker in several neoplasms. In the present study we aimed to examine whether RKIP expression is associated with GIST clinical–pathological features. Using immunohistochemistry, we determined RKIP expression levels in a well-characterised series of 70 GISTs. We found that RKIP is expressed in the great majority of cases, and absent in approximately 9% of GISTs. Additionally, we found that loss of RKIP expression was not due to the promoter methylation as assessed by methylation-specific PCR. Loss of RKIP expression was associated with poor disease-specific survival and with tumour necrosis in GISTs. Furthermore, a statistical tendency was observed between the positive RKIP expression and absence of metastasis. So far, this is the first study assessing RKIP expression levels in GISTs. We conclude that loss of RKIP expression could have an important role as prognostic marker in GISTs.     

High PITX1 expression in lung adenocarcinoma patients is associated with DNA methylation and poor prognosis

Background

Pituitary homeobox 1 (PITX1) is a member of the PITX gene family which is vital to proper development of early embryo. However, the relationship of PITX1 expression and overall survival (OS) in non-small cell lung cancer (NSCLC) is not clear.

Aberrant deleted in liver cancer‐1 expression is associated with tumor metastasis and poor prognosis in urothelial carcinoma

This study explored the potential role of deleted in liver cancer‐1 (DLC‐1) as a prognostic indicator of cancer metastasis and survival in urothelial carcinoma (UC). Tissue microarrays were constructed from paraffin‐embedded specimens from 88 UC patients, and immunohistochemical staining was performed to investigate the association of DLC‐1 with clinicopathologic characteristics and clinical outcome. The DLC‐1 expression showed a significant positive correlation with tumor location (p = 0.041) and a significant negative correlation with advanced histological grade (p = 0.013). In tumors with low DLC‐1 expression, Bcl‐2 positivity was observed in 24.4% of cases. The DLC‐1 expression had significant negative associations with Bcl‐2 expression (p = 0.032) and with highly metastatic UC (p = 0.032). Kaplan–Meier analysis showed that DLC‐1 protein expression was negatively associated with both overall survival (OS) (p = 0.035) and with distant metastasis‐free survival (DMFS) (p = 0.041), but not with disease‐free survival. Multivariate analyses indicated that tumor size was the significant independent predictors of OS (p = 0.048); however, only DLC‐1 expression was a significant independent predictor of DMFS (p = 0.019). In conclusion, reduced DLC‐1 protein expression may be an important factor in tumor progression and a useful prognostic molecular marker in UC.     

Reduced FHIT expression is associated with tumor progression in sporadic colon adenocarcinoma

Purpose

Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma.

Methods

The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis.

Results

One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10^− 6). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant.

Conclusions

Our overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors.     

Decreased expression of SEMA3A is associated with poor prognosis in gastric carcinoma

Background/purpose: SEMA3A (semaphorin-3A), is a secreted protein that belongs to the semaphorin family and can function as both a chemoattractive agent or a chemorepulsive agent. SEMA3A has been shown to be a tumor suppressor in various cancers. This study investigated the expression of SEMA3A in gastric cancer and its prognostic value for gastric cancer patients. Methods: We examined the expression of SEMA3A in paired cancerous and matched adjacent noncancerous gastric mucosa tissues by real-time quantitative RT-PCR (qRT-PCR) and western blotting. In vitro, we evaluate the effects of SEMA3A on gastric cancer cell proliferation and migration by MTT, transwell and wound-healing assays. Furthermore, we analyzed SEMA3A expression in 128 patients who underwent resection procedures using immunohistochemistry. The relationships between the SEMA3A expression levels, the clinicopathological factors, and patient survival were investigated. Results: Our results revealed decreased SEMA3A mRNA (P = 0.0037) and protein (P = 0.033) expression in tumor tissue samples compared with matched adjacent non-tumorous tissue samples. Overexpression of SEMA3A inhibits gastric cancer cell proliferation and migration in vitro. Immunohistochemical staining data showed that SEMA3A expression was significantly decreased in 54.68% of gastric cancer cases. In addition, the chi-square test revealed that low SEMA3A expression was significantly correlated with poor differentiation (P = 0.015), Vascular invasion (P = 0.001), depth of invasion (P < 0.001), lymph node metastasis (P = 0.029), distant metastasis (P = 0.002) and advanced TNM stage (P = 0.003). SEMA3A expression was positively correlated with clinical TNM stage, that suggested the more advanced clinical TNM stage corresponding to the lower expression level of SEMA3A (r_s = -0.322, P < 0.001) by Spearman rank correlation analysis. Kaplan-Meier survival analysis demonstrated that low expression of SEMA3A was significantly correlated with a poor prognosis for gastric cancer patients (P < 0.001). The multivariate analysis revealed that SEMA3A expression was an independent prognostic factor of the overall survival rate of patients with gastric cancer. Conclusion: SEMA3A expression decreased significantly as gastric cancer progressed and metastasized, suggesting that SEMA3A might serve as a candidate tumor suppressor and a potential prognostic biomarker in gastric carcinogenesis.     

Low RKIP expression associates with poor prognosis in bladder cancer patients

Urothelial bladder cancer (UBC) is a heterogeneous type of disease. It is urgent to screen biomarkers of tumour aggressiveness in order to clarify the clinical behaviour and to personalize therapy in UBC patients. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor, and its downregulation is associated with metastatic events in an increasing number of solid tumours. We evaluated the clinical and prognostic significance of RKIP expression in patients with high risk of progression UBC. Using immunohistochemistry, we determined RKIP expression levels in a series of 81 patients with high-grade pT1/pTis or muscle-invasive UBC. Staining of CD31 and D2-40 was used to assess blood and lymphatic vessels, in order to distinguish between blood and lymphatic vessel invasion (LVI). We found that 90 % of pT1/pTis tumours, 94 % of non-muscle invasive papillary tumours and 76 % of the cases without LVI occurrence expressed RKIP in >10 % of cells. In this group, we observed a subgroup of tumours (42 %) in which the tumour centre was significantly more intensely stained than the invasion front. This heterogeneous pattern was observed in 63 % of the cases with LVI. Low RKIP expression was associated with poorer 5-year disease-free and overall survival rates, and remained as an independent prognostic factor for disease-free survival. Loss of RKIP expression may be an important prognostic factor for patients with high risk of progression bladder cancer.     

Increased MT2-MMP expression in gastric cancer patients is associated with poor prognosis

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that contribute to tumorigenesis and metastasis due to their ability to degrade the extracellular matrix (ECM) and basement membrane. In despite of many reports in other solid tumors, the role of membrane type-2 MMP (MT2-MMP) in gastric cancer (GC) remains to be elucidated. The aim of this study was to investigate MT2-MMP expression in human GC tissue microarray (TMA) samples using immunohistochemistry (IHC). We found that MT2-MMP expression in tumor tissues was significantly higher compared to peritumoral tissues (P < 0.01). However, there were no statistically significant differences between MT2-MMP expression and clinicopathological parameters. In addition, univariate and multivariate Cox regression analysis showed GC patients with high MT2-MMP expression have poor overall survival (OS) compared to patients with low MT2-MMP expression (P = 0.013, P = 0.040, respectively). In conclusion, MT2-MMP is involved in GC invasion and metastasis and may serve as an independent prognostic factor for GC patients.     

High RSF-1 expression correlates with poor prognosis in patients with gastric adenocarcinoma

Aim

To investigate the expression and prognostic significance of RSF-1 in gastric adenocarcinoma.

Methods

RSF-1 expression was analyzed using immunohistochemical staining on tissue samples from a consecutive series of 287 gastric adenocarcinoma patients who underwent tumor resections between 2003 and 2006.The relationship between RSF-1 expression, clinicopathological factors, and patient survival was investigated.

Results

Immunohistochemical staining indicated that RSF-1 is highly expressed in 52.6% of gastric adenocarcinomas. RSF-1 expression levels were closely associated with tumor size, histological differentiation, tumor stage, and lymph node involvement. Kaplan-Meier survival analysis showed that high RSF-1 expression exhibited a significant correlation with poor prognosis for gastric adenocarcinoma patients. Multivariate analysis revealed that RSF-1 expression is an independent prognostic parameter for the overall survival rate of gastric adenocarcinoma patients.

Conclusion

Our data suggest that RSF-1 plays an important role in gastric adenocarcinoma progression and that high RSF-1 expression predicts an unfavorable prognosis in gastric adenocarcinoma patients.     

Over-expression of lncRNA DANCR is associated with advanced tumor progression and poor prognosis in patients with colorectal cancer

Despite advances made in the diagnosis and treatment of human colorectal cancer (CRC), the long-term survival for CRC remains poor. Long non-coding RNA anti-differentiation ncRNA (lncRNA DANCR) was identified to be involved in carcinogenesis of hepatocellular carcinoma. While its expression in CRC and potential role in tumor progression is still unknown. In the present study, we investigated the expression level of lncRNA DANCR as well as its association with CRC progression and prognosis. The expression of lncRNA DANCR was detected by quantitative real-time PCR (qRT-PCR) in 104 CRC specimens. The prognostic value of lncRNA DANCR was further analysis. Our results showed that lncRNA DANCR expression was increased in CRC tissues compared with that in adjacent normal tissues (P<0.05). In addition, tumors with high lncRNA DANCR expression was correlated with TNM stage, histologic grade, and lymph node metastasis (P<0.05). Kaplan-Meier analysis showed that patients with high lncRNA DANCR expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with the low lncRNA DANCR expression group (P<0.05). Moreover, in a multivariate Cox model, our results showed that lncRNA DANCR expression was an independent poor prognostic factor for both OS and DFS in CRC. Our data indicated that lncRNA DANCR expression might be a novel potential biomarker for CRC prognosis.     

Expression of Cbl linking with the epidermal growth factor receptor system is associated with tumor progression and poor prognosis of human gastric carcinoma

Cbl proteins play important roles in downregulation of growth factor receptors by acting as ubiquitin ligases and multi-adapter proteins. Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) has been shown to be controlled by Cbl. In the present study, we examined the expression of Cbl in gastric carcinomas and studied the correlation of Cbl expression with clinicopathological characteristics as well as EGFR expression. Cbl protein was expressed in 67% (82/122) of gastric carcinomas, and diffuse expression of Cbl was detected in 29% (35/122) of the cases. The incidence of cases with diffuse expression of Cbl was significantly higher in advanced cases (28/70, 40%) than in early cases (7/52, 14%) (P=0.0010). Diffuse expression of Cbl was significantly associated with metastasis of tumor cells in lymph nodes (P=0.0318). Diffuse expression of EGFR was significantly associated with depth of invasion (P=0.0057), lymph-node metastasis (P=0.0371) and tumor stages (P=0.0278). As the grades of Cbl expression became stronger, the cases with diffuse EGFR expression increased, the positive correlation being significant (P=0.049). All the cases with diffuse expression of Cbl and EGFR were found to show nodal metastasis and to be at an advanced stage. Moreover, the prognosis of the patients with synchronous diffuse expression of Cbl and EGFR was significantly poorer than that of the patients negative for Cbl and focal or negative for EGFR (P=0.0086). The expression of Cbl protein was clearly induced in gastric carcinoma cell lines by transforming growth factor- treatment. These results suggest that Cbl in connection with the EGFR system may be associated with stomach carcinogenesis, invasion and metastasis. Cbl may serve as a novel molecular marker for aggressive gastric carcinoma.     

Overexpression of EZH2 and loss of expression of PTEN is associated with invasion, metastasis, and poor progression of gallbladder adenocarcinoma

Overexpression of EZH2 and inactivation or loss of PTEN expression was observed in invasive and metastatic tumors. However, their expressions and clinical significances in gallbladder cancer (GBC) have rarely been reported. In this study, we investigated EZH2 and PTEN expression in an extensive collection of human gallbladder cancer samples and benign lesions of gallbladder using immunohistochemistry. Overexpression of EZH2 was detected in 53.7% of gallbladder adenocarcinomas associated with poor differentiation, lymph node metastasis, and invasion, while loss of PTEN expression was identified in 51.8% of adenocarcinomas with high grade, metastatic, and invasive tumors. Univariate Kaplan-Meier analysis showed that overexpression of EZH2 (p = 0.013) and loss of PTEN expression (p = 0.008) were significantly associated with decreased overall survival. Multivariate Cox regression analysis revealed that overexpression of EZH2 (p = 0.011) or loss of PTEN expression (p = 0.009) is a predictor of poor prognosis in gallbladder adenocarcinoma. Our study suggests that overexpression of EZH2 and loss of PTEN expression might be closely related to the carcinogenesis, progression, clinical biological behavior, and prognosis of gallbladder adenocarcinoma.     

High expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinoma is associated with tumor invasion and poor prognosis

The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which – like RasGRF2 – is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.     

Aberrant methylation and loss of expression of O-methylguanine-DNA methyltransferase in pulmonary squamous cell carcinoma and adenocarcinoma

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells against the carcinogenic effects of alkylating agents. The methylation status of the MGMT gene was investigated by methylation-specific polymerase chain reaction (PCR) and expression status was investigated by immunohistochemistry in 70 cases of pulmonary squamous cell carcinoma (pulmonary SqCC), including 23 cases of the central type and 47 cases of the peripheral type, and in 53 cases of the peripheral type of pulmonary adenocarcinoma (AC). The frequency of MGMT methylation was 36% in SqCC and 42% in AC. Cases with MGMT methylation correlated significantly with T factor in SqCC (P = 0.047) and AC (P = 0.03). In SqCC, the frequency of MGMT methylation was 26% in the central type and 40% in the peripheral type; a significant correlation was not found (P = 0.29). In AC with mixed subtypes showing MGMT methylation, the level of MGMT expression in the bronchioloalveolar carcinoma (BAC) area (non-invasive status) was significantly higher than that in the papillary or acinar AC area (invasive status; P = 0.0002). This trend was not found in AC with mixed subtypes showing no MGMT methylation (P = 0.10). These findings suggest that MGMT inactivation is an event that occurs in the late carcinogenic process in SqCC and AC, and that AC progress from non-invasive status to invasive status with MGMT inactivation induced by the promoter DNA methylation.     

High expression of Twist is associated with tumor aggressiveness and poor prognosis in patients with renal cell carcinoma

Aims: Twist has been reported to play crucial roles for malignant aggressiveness; however, detailed pathological significance of Twist in renal cell carcinoma (RCC) is not fully understood. The present study was to clarify clinical significance and molecular functions of Twist in patients with RCC. Methods: Twist expression was examined by immunohistochemical techniques in 156 formalin-fixed specimens. Cell proliferation, angiogenesis, and apoptosis were measured as the percentage of Ki-67-positive cells (proliferation index, PI), CD31-stained vessels (microvessel density, MVD), and TUNEL-positive cells (apoptotic index, AI). In addition, semi-quantification of matrix metalloproteinase (MMP)-2 was performed. Macrophages were identified with anti-CD68 antibody, and the tumor associated macrophage (TAM) density was calculated as CD68-positive cells per high-power field. Results: Twist expression was positively associated with grade, pT stage, and metastasis (p<0.001). We also noticed that its expression was considerably higher in cancer cells of sarcomatoid RCC and in those at the edge of the tumors. Twist expression was positively correlated with PI, MVD, MMP2 expression, and TAM density (P<0.001), but not with AI, and MMP-2 expression and TAM density were independently correlate by multi-variate analyses. Kaplan-Meir survival curves showed high Twist expression was a worse predictor for cause-specific survival (P<0.001). Conclusions: Twist plays important roles in tumor growth, progression, and survival in patients with RCC patients. Such pathological mechanisms are significantly associated with increased cancer cell proliferation, angiogenesis, MMP2 expression, and macrophage recruitment. These findings are important information for discussion of treatment and observation strategies in these patients.