Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma

The expression of the Wilms' tumor gene WT1 was examined by immunohistochemistry in 40 cases of pancreatic ductal adenocarcinoma. WT1 protein was expressed in 30 (75%) of the 40 pancreatic ductal adenocarcinomas, but not in the remaining 10 (25%). In normal pancreatic ductal cells, WT1 protein was undetectable. No correlations between WT1 expression and clinicopathological parameters such as age, sex, T or N stage, tumor location, and tumor differentiation were observed. Treatment with WT1 antisense oligomers significantly inhibited the growth of five human pancreatic cancer cell lines, PSN1, MiaPaCa2, ASPC1, BxPC3, and PCI6, expressing the WT1 gene. These results indicate an important role of the WT1 gene in the tumorigenesis of pancreatic ductal adenocarcinoma expressing WT1 and provide a rationale for new treatment strategies to treat pancreatic ductal adenocarcinoma by targeting the WT1 gene and its product.     

Overexpression of rhophilin 2 promotes pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is the seventh leading cause of global cancer deaths. In recent years, targeted therapy has been used for pancreatic cancer; however, the drugs available for use in targeted therapy for pancreatic cancer are still very limited. Hence, identification of novel targeted molecules for PDAC is required. Rhophilin 2 (RHPN2) was proven to be a driver gene in glioblastoma. However, the function of RHPN2 in PDAC remains unknown. In the present study, the function of RHPN2 was investigated. The RHPN2 levels were overexpressed by pcDNA3.1-RHPN2 and downregulated by si-RHPN2. Cell proliferation was assessed using the MTT assay and apoptosis was assessed using flow cytometry. The results revealed that high RHPN2 levels in PDAC tissue were correlated with a low overall survival rate of patients with PDAC. Inhibition of RHPN2 reduced SW1990 and PANC1 proliferation and increased the rate of apoptosis. Network analysis demonstrated that centrosomal protein 78 expression was negatively correlated with RHPN2 expression. In conclusion, the present study demonstrated that RHPN2 may promote PDAC making it a potential candidate for targeted therapy.     

Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas

Pancreatic ductal adenocarcinoma (PDA) remains a devastating disease with nearly equal incidence and mortality rates. Over the past few decades, a litany of randomized clinical trials has failed to improve the outcome of this disease. More recently, the combination chemotherapy regimen FOLFIRINOX has shown improvement in overall survival over the single agent gemcitabine, and nab-paclitaxel (an albumin-coated formulation of paclitaxel) in combination with gemcitabine has shown promising results in phase II studies. Despite limited impact on patient care as of yet, the molecular and biologic understanding of PDA has advanced substantially. This includes understanding the genomic complexity of the disease, the potential importance of the tumor microenvironment, the metabolic adaptation of PDA cells to obtain nutrients in a hypoxic environment, and the role of pancreatic cancer stem cells. These fundamental discoveries are starting to be translated into clinical studies. In this overview, we discuss the implications of biologic understanding of PDA in clinical research and provide insights for future development of novel approaches and agents in this disease.     

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.     

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

     

Clinicopathological significance of galectin-3 expression in ductal adenocarcinoma of the pancreas.

PURPOSE: Galectin-3, a member of the beta-galactoside-binding lectin family, has multiple biological functions including cell-cell interactions and cell-extracellular matrix adhesion, cellular proliferation, cellular differentiation, and apoptosis. The aim of this study was to determine the relationship of galectin-3 expression to clinicopathological findings and patient prognosis in ductal adenocarcinoma of the pancreas. EXPERIMENTAL DESIGN: We examined galectin-3 expression in 104 surgically resected pancreatic ductal adenocarcinoma cases with stages I through IV using immunohistochemistry and investigated the relationship of it to overall survival. RESULTS: Patients were divided into two groups: a low expression group, where <60% of tumor cells were positive; and a high expression group, where > or =60% of tumor cells were positive. Cases in the low expression group had a significant tendency to be at later stages, to have distant metastasis, and to have less differentiated tumors, compared with cases in the high expression group (P = 0.001 for stage, P = 0.001 for metastasis, and P = 0.006 for differentiation). Postoperative overall survival was worse in the low galectin-3 expression group than in the high galectin-3 expression group (P = 0.004). Multivariate analysis showed that the risk ratio of prognosis was 2.06 among patients in the low galectin-3 expression group compared with the high galectin-3 expression group (P = 0.006). CONCLUSIONS: Decreased expression of galectin-3 was associated with advanced stage, tumor de-differentiation, and metastasis in ductal adenocarcinoma of the pancreas. Galectin-3 expression might be a useful prognostic marker for survival in ductal adenocarcinoma of the pancreas.     

Overexpression of cellular iron import proteins is associated with malignant progression of esophageal adenocarcinoma.

PURPOSE: There is growing evidence that iron is important in esophageal adenocarcinoma, a cancer whose incidence is rising faster than any other in the Western world. However, how iron mediates carcinogenesis at the molecular level remains unclear. In this study, we investigated the expression of iron transport proteins involved in cellular iron import, export, and storage in the premalignant lesion Barrett's metaplasia and esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Perls' staining was used to examine iron deposition in tissue. mRNA expression in samples of Barrett's metaplasia matched with esophageal adenocarcinoma and samples of Barrett's metaplasia without evidence of adenocarcinoma were examined by real-time PCR. Semiquantitative immunohistochemistry was used to examine cellular localization and protein levels. The effect of iron loading on cellular proliferation and iron transporter expression was determined in esophageal cell lines OE33 and SEG-1 using a bromodeoxyuridine assay and real-time PCR, respectively. RESULTS: In the progression of Barrett's metaplasia to adenocarcinoma, there was overexpression of divalent metal transporter 1 (DMT1), transferrin receptor 1, duodenal cytochrome b, ferroportin, and H-ferritin, and these changes were associated with increased iron deposition. Overexpression of DMT1 was further associated with metastatic adenocarcinoma. Iron loading OE33 and SEG-1 cells caused increased cellular proliferation, which was associated with increased H-ferritin and decreased transferrin receptor 1 and DMT1 expression. CONCLUSIONS: Progression to adenocarcinoma is associated with increased expression of iron import proteins. These events culminate in increased intracellular iron and cellular proliferation. This may represent a novel mechanism of esophageal carcinogenesis.     

Tumour characteristics predictive of survival following resection for ductal adenocarcinoma of the head of pancreas.

AIMS: We have maintained a highly conservative policy in selecting patients with carcinoma of the head of pancreas for resection. This has been based on tumour size, evidence of lymph node involvement or local invasion outside of the gland at laparotomy, laparoscopy or CT imaging. This study investigated our survival rates following pancreatic resection and examined clinicopathological predictors of survival. METHODS: Sixty-two consecutive patients undergoing pancreatic resections for malignancy were identified from 1999 onwards. Thirty-three underwent resection for pancreatic ductal adenocarcinoma and were included in our analysis, the remainder included resections for ampullary adenocarcinoma (n=20) or other malignancies (n=9). Patient, tumour and operative characteristics were analysed to assess predictors of survival following resection (Kaplan-Meier survival curves). RESULTS: Median survival following resection for ductal pancreatic adenocarcinoma was 54 months (ampullary adenocarcinomas achieved a median survival of 62 months) and thirty-day mortality was 2.7% (n=1). Survival was not associated with any demographic or intraoperative factors, such as blood loss, operative duration or anaesthetic technique. Survival curves were significantly worse when perineural or vascular invasion was evident histologically (p=0.023 and 0.0023 respectively). Patients with positive lymph nodes had a significantly shorter survival (p=0.0030) especially when lymph node status was expressed as a percentage of total lymph node yield. If more than 20% of retrieved lymph nodes were positive for tumour, this was a clear predictor of survival (p<0.0001). A positive resection margin was also associated with shortened survival (p=0.0291). CONCLUSION: Despite the advances made in the management of pancreatic cancer, tumour biology still dictates long-term survival. A highly selective surgical approach to the management of these patients results in good long-term survival.     

Rare ductal adenocarcinoma of the pancreas in patients younger than age 40 years

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are extremely rare before age 40 years. The objective of the current study was to determine whether the features of PDACs in patients age < 40 years differ from those in older patients. The authors reviewed the literature and their own files. METHODS: The cases reported in the literature were evaluated to determine their precise diagnoses and characteristic features. In a series of 439 PDACs from the authors' files, tumors in patients age < 40 years were identified, and their clinicopathologic features and certain genetic features were compared with those in a selected group of patients age > 40 years. RESULTS: Of 71 pancreatic carcinomas reported in patients age < 40 years, only 20 fully qualified as PDACs. The remaining tumors represented malignancies other than PDACs, such as pancreatoblastoma, solid-pseudopapillary neoplasms, acinar cell carcinomas, and endocrine tumors. PDACs in patients age < 20 years were the absolute exception and commonly were associated with risk factors such as Peutz-Jeghers syndrome, hereditary pancreatic cancer syndrome, and preceding radiotherapy. In the authors' series of patients, there were 6 PDACs and 4 PDAC variants in patients age < 40 years (0.2%), all in male patients. These tumors compared well with the PDACs in patients age > 40 years in their pathologic and molecular findings. Three patients were age < or = 20 years, and 2 of those patients had a mucinous component with MUC2 positivity. CONCLUSIONS: The incidence of PDACs in patients age < 40 years was approximately 0.3%, and the incidence in patients age < 20 years was 0.1%. Their clinicopathologic findings were comparable to those in patients age > 40 years, but they seemed to include more variants, particularly mucinous carcinomas. In addition, PDACs in younger patients frequently appeared to be associated with genetic factors.     

Factors influencing survival after resection for ductal adenocarcinoma of the head of the pancreas

BACKGROUND AND OBJECTIVES: Recent reports have demonstrated improvement in the 5-year actuarial survival for patients with resected ductal adenocarcinoma. The purpose of this study is to determine the factors favoring long-term survival after pancreaticoduodenectomy. METHODS: Between 1974 and 1995, 75 patients with pancreatic head carcinoma underwent pancreaticoduodenectomy in our department. RESULTS: Overall postoperative mortality rate was 5. 3% and morbidity was 24%. Median survival following resection was 17 months. Estimated 1-, 2-, and 5-year survival rates were 68%, 46.7%, and 18.7%, respectively. Five-year survival was greater for node-negative than for node-positive patients (41.7% vs. 7.8%, P < 0. 001) and for smaller (<3 cm) than for larger tumors (33.3% vs. 8.8%, P < 0.006). The 5-year survival in patients with negative margins (n = 60) was 23.3%, whereas no patient with positive margins (n = 15) survived at 13 months (P < 0.001). Multivariate analysis, performed by the Cox proportional hazards model, indicated that margin status, lymph node metastasis, tumor size, and poor histological differentiation were independent predictors of poor survival. CONCLUSIONS: Five-year survival for patients undergoing pancreaticoduodenectomy for ductal adenocarcinoma of the pancreas was 18.7%. Survival was greater in the group of patients with negative lymph nodes, tumor size <3 cm, and negative margin status.     

Overexpression of Necl‐5 correlates with unfavorable prognosis in patients with lung adenocarcinoma

Nectin‐like molecule‐5 (Necl‐5) is an immunoglobulin (Ig)‐like molecule that is up‐regulated in many types of cancer cells. It was shown experimentally that Necl‐5 enhances cell migration, proliferation, and metastasis, but its clinical significance has not been documented. The aim of this study was to observe the expression of Necl‐5 in surgically resected primary lung adenocarcinomas and to investigate its clinical significance. A total of 63 surgically resected primary pulmonary adenocarcinoma tissues were investigated by immunohistochemistry for the expression of Necl‐5. The relationship between expression of Necl‐5 and clinicopathological features was analyzed, and the influence of Necl‐5 expression on outcomes in these patients was assessed. A strong expression of Necl‐5 by cancer cells was observed in 43 of the 63 tumors. The overexpression of Necl‐5 by cancer cells was significantly associated with lymph node metastasis (P = 0.0398), TNM staging (P = 0.0367), and the bronchioloalveolar carcinoma ratio of tumors (P = 0.0423). Furthermore, the disease‐free survival rate in patients with positive Necl‐5 overexpression was significantly lower than that in patients with negative Necl‐5 overexpression (P = 0.0004). Multivariate survival analysis revealed Necl‐5 expression to be an independent risk factor for an unfavorable outcome (P = 0.0294). Additionally, an analysis including only the stage I cases revealed that the disease‐free survival rate of the Necl‐5‐positive group was significantly lower than that of the Necl‐5‐negative group (P = 0.0192). These results indicate that Necl‐5 plays a role in mediating tumor cell invasion and that the overexpression of Necl‐5 in cancer cells has clinical significance for prognostic evaluation of patients with primary pulmonary adenocarcinoma. (Cancer Sci 2010; 101: 1326–1330)     

Overexpression of SPAG9 correlates with poor prognosis and tumor progression in hepatocellular carcinoma

Sperm-associated antigen 9 (SPAG9) was reported as a novel biomarker for several cancers and associated with the malignant behavior of cancer cells. However, its expression pattern and biological role in human hepatocellular carcinoma (HCC) have not been reported. In the present study, we analyzed SPAG9 expression in human HCC tissues by immunohistochemistry and found that SPAG9 overexpression is correlated with tumor stage (p?p?=?0.019), tumor size (p?=?0.034), AFP levels (p?=?0.006), and tumor relapse (p?=?0.0017). Furthermore, SPAG9 overexpression is correlated with poor overall survival (p?p?=?0.002). Transfection of SPAG9 small interfering RNA (siRNA) was performed in Bel-7402 cell line. Colony formation and MTT showed that SPAG9 siRNA knockdown inhibited HCC cell proliferation. We also found that SPAG9 depletion could increase cell apoptosis. In addition, the level of cyclin D1 and cyclin E protein expression was downregulated after siRNA treatment. In conclusion, SPAG9 is overexpressed in human HCC and serves as a prognostic marker. SPAG9 contributes to cancer cell growth through regulation of cyclin proteins.     

Aberrant DNA methyltransferase expression in pancreatic ductal adenocarcinoma development and progression

Background

Altered gene methylation, regulated by DNA methyltransferases (DNMT) 1, 3a and 3b, contributes to tumorigenesis. However, the role of DNMT in pancreatic ductal adenocarcinoma (PDAC) remains unknown.

Methods

Expression of DNMT 1, 3a and 3b was detected in 88 Pancreatic ductal adenocarcinoma (PDAC) and 10 normal tissue samples by immunohistochemistry. Changes in cell viability, cell cycle distribution, and apoptosis of PDAC cell lines (Panc-1 and SW1990) were assessed after transfection with DNMT1 and 3b siRNA. Levels of CDKN1A, Bcl-2 and Bax mRNA were assessed by qRT-PCR, and methylation of the Bax gene promoter was assayed by methylation-specific PCR (MSP).

Results

DNMT1, 3a and 3b proteins were expressed in 46.6%, 23.9%, and 77.3% of PDAC tissues, respectively, but were not expressed in normal pancreatic tissues. There was a co-presence of DNMT3a and DNMT3b expression and an association of DNMT1 expression with alcohol consumption and poor overall survival. Moreover, knockdown of DNMT1 and DNMT3b expression significantly inhibited PDAC cell viability, decreased S-phase but increased G1-phase of the cell cycle, and induced apoptosis. Molecularly, expression of CDKN1A and Bax mRNA was upregulated, and the Bax gene promoter was demethylated. However, a synergistic effect of combined DNMT1 and 3b knockdown was not observed.

Conclusion

Expression of DNMT1, 3a and 3b proteins is increased in PDAC tissues, and DNMT1 expression is associated with poor prognosis of patients. Knockdown of DNMT1 and 3b expression arrests tumor cells at the G1 phase of the cell cycle and induces apoptosis. The data suggest that DNMT knockdown may be a novel treatment strategy for PDAC.     

RhoC基因的过量表达与肝细胞癌的侵袭转移

目的　研究探讨RhoC基因mRNA和蛋白的过量表达与肝细胞癌 (HCC)的发生发展和侵袭转移的关系。方法　采用逆转录聚合酶链反应 (RT PCR)和Western印迹法 ,检测 2 5例HCC及其癌旁肝组织、4例门静脉主干癌栓或肝外转移灶中RhoCmRNA和蛋白的表达 ;同时采用PCR产物单链构象多态性 (PCR SSCP)银染法检测RhoC基因的突变情况。结果　HCC组织和癌旁肝组织中均可检测到RhoC基因mRNA和蛋白的表达 ,RhoCmRNA在HCC组织中的表达较癌旁肝组织高 ,其吸光度(A)比值分别为 1.8± 1.1和 1.0± 0 .7,差异有显著性 (P <0 .0 1) ;RhoC蛋白在HCC组织中的表达同样高于癌旁肝组织 ,其A比值分别为 33992± 10 384和 17342± 9998,差异有显著性 (P <0 .0 1)。 4例门静脉主干癌栓或肝外转移灶中的RhoC基因mRNA和蛋白的表达量分别为 3.3± 0 .5和 6 386 5±9116 ,较HCC肝内病灶高 ,差异有显著性 (P <0 .0 1) ;RhoCmRNA和蛋白在分化较差、结节数较多、有静脉浸润或伴有肝外转移灶的HCC中过量表达 (P <0 .0 5 )。全部HCC经PCR SSCP银染法分析均未发现异常泳动条带。结论　RhoC基因的过量表达与HCC的发生发展和侵袭转移密切相关 ,且可能是通过过量表达而非突变发挥作用。     

Clinicopathological significance and molecular regulation of maspin expression in ductal adenocarcinoma of the pancreas

We evaluated the biological relevance of maspin expression in pancreatic ductal adenocarcinoma and studied regulatory mechanisms of maspin gene activation in pancreatic carcinoma cell lines. Maspin expression was immunohistochemically detected in a series of 57 pancreatic ductal adenocarcinomas, 51 (90%) of which were classified as high-expressers. In lymph node metastases, maspin expression was somewhat decreasingly found in 39/49 (80%). Maspin high-expressers showed predominantly a low histological grade (p=0.013). Moreover, maspin expression was found in two mixed ductal-endocrine carcinomas, but not in 10 endocrine tumors and the surrounding normal pancreatic tissues. Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive pancreatic cancer cell lines as well as maspin-negative PANC-1 cells. Additionally, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin mRNA in PANC-1 cells. Our results indicate that maspin expression is up-regulated in most if not all pancreatic ductal adenocarcinomas and may be related to the development and differentiation, and that DNA methylation and histone deacetylation may suppress maspin gene activation in pancreatic cancer cells.