Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer.

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G0/G1 phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.

Members of the EpCAM signalling pathway are expressed in gastric cancer tissue and are correlated with patient prognosis.

BACKGROUND: We investigated the expression of members of the epithelial cell adhesion molecule (EpCAM) signalling pathway in gastric cancer (GC) testing the following hypotheses: are these molecules expressed in GC and are they putatively involved in GC biology. METHODS: The study cohort consisted of 482 patients. The following members of the EpCAM signalling pathway were analysed by immunohistochemistry and were correlated with various clinico-pathological patient characteristics: extracellular domain of EpCAM (EpEX), intracellular domain of EpCAM (EpICD), E-cadherin, ß-catenin, presenilin-2 (PSEN2), and ADAM17. RESULTS: All members of the EpCAM signalling pathway were differentially expressed in GC. The expression correlated significantly with tumour type (EpEX, EpICD, E-cadherin, ß-catenin, and PSEN2), mucin phenotype (EpEX, EpICD, ß-catenin, and ADAM17), T-category (EpEX, E-cadherin, and ß-catenin), N-category (EpEX and ß-catenin), UICC tumour stage (EpEX, EpICD, ß-catenin, and PSEN2), tumour grade (EpEX, EpICD, E-cadherin, ß-catenin, and PSEN2), and patients' survival (EpEX, EpICD, and PSEN2). A significant coincidental expression in GC was found for EpEX, EpICD, E-cadherin, ß-catenin, PSEN2, and ADAM17. Decreased immunodetection of EpEX in locally advanced GC was not associated with decreased EpCAM mRNA levels. CONCLUSION: All members of the EpCAM signalling pathway are expressed in GC. The expression correlated significantly with each other and with various clinico-pathological patient characteristics, including patients' survival. Thus, the EpCAM signalling pathway is a highly interesting putative therapeutic target in GC.

WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells.

Resistance to chemotherapy is a major obstacle for curative treatment of human gastric cancer (GC). However, the underlying molecular mechanisms are largely unknown. Wingless-type MMTV integration site family members (WNTs) are secreted glycoproteins involved in embryogenesis and, on inappropriate expression in the adult, in cancer. Here, we show expression of WNT6 in GC patient specimens, human GC cell lines and in a mouse model of GC. In human GC cells, WNT6 expression was enhanced by caveolin-1 (Cav1), a scaffold protein of plasma membrane caveolae. WNT6 knock-down and overexpression experiments demonstrated that WNT6 increased the resistance to apoptotic cell death induced by the anthracycline chemotherapeutics epirubicin (Epi) and doxorubicin (Dox). Epi increased the activity of the human WNT6 promoter through Cav1-dependent binding of ß-catenin to the proximal WNT6 promoter. Epi increased both WNT6/Wnt6 and Cav1 expression in human GC cells and within the tumor area of a murine model of GC (CEA424-SV40 TAg). In GC patients, WNT6 expression was positively associated with the tumor stage and the nodal status, and inversely correlated with the response to ECF (Epi, cisplatin, 5-fluorouracil) chemotherapy. These results showed that WNT6 and Cav1 are upregulated by chemotherapeutics and enhance the resistance of GC cells to anthracycline drugs. Understanding the molecular mechanisms driving WNT6/Cav1-induced drug resistance will provide benefits in developing new therapies for GC.

Her2/neu testing in gastric cancer: evaluating the risk of sampling errors.

BACKGROUND: We evaluated the risk of sampling errors in specimens of biopsy size, which may be caused by heterogeneous overexpression of Her2/neu in gastric cancer (GC). PATIENTS AND METHODS: The study cohort comprised 454 gastrectomy patients with adenocarcinoma of the stomach or esophago-gastric junction. Tissue micro-arrays (TMAs) served as 'biopsy procedure' and were generated from formalin-fixed and paraffin-embedded tissue: five tissue cylinders were collected randomly from each tumor, rendering 2230 core cylinders. These were compared with 454 whole tissue sections obtained from the same paraffin blocks. Her2/neu expression and gene amplification were analyzed by immunohistochemistry and in situ hybridization. The Her2/neu status was determined according to GC scoring system by two independent observers. RESULTS: In whole tissue sections, 37 (8.1%; observer 1) and 38 (8.4%; observer 2) of the GCs, and in the corresponding TMAs, 28 (6.3%; observer 1) and 28 (6.3%; observer 2) of the GCs were classified as Her2/neu-positive (kappa value 98.5% and 96.2%; P < 0001). Comparison of whole tissue sections with corresponding TMAs showed a false-negative rate of 24% and a false-positive rate of 3% for TMAs. CONCLUSION: Assessment of the Her2/neu status in tissue biopsies carries a significant risk of sampling errors, thereby rendering patients unsuitable for treatment with trastuzumab.

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

BACKGROUND: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis. METHODS: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro. RESULTS: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar. CONCLUSION: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer.

BACKGROUND: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC). METHODS: Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT-PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association. RESULTS: Both real-time PCR-based expression arrays and qRT-PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues. CONCLUSION: Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

[New approaches to early detection, estimation of prognosis and therapy for malignant tumours of the gastrointestinal tract]. / Neue Wege zur Früherkennung, Prognoseabschätzung und Therapie maligner Tumoren des Gastrointestinaltraktes.

Tumours of the gastrointestinal tract are the single largest group of malignant tumours of humans accounting for approximately 2 million new cases and over 1.2 million deaths annually. They are most commonly a disease of the elderly, and their relative and absolute frequency will rise continuously due to an ageing population. This explains why the prevalence of gastric cancer increases in spite of a decreasing incidence. Gastrointestinal tumours will remain a major clinical and health economical challenge. The prognosis is often dismal. Most of these cancers are diagnosed in an advanced stage, which prohibits curative treatment and limits treatment options. In order to circumvent these problems, we need to develop strategies that allow identification of at-risk patients and tumours at an early stage, and raise the compliance in the general population for screening programmes. There is a great need in clinics for prognostic and predictive biomarkers that are able to tailor patient treatment at different stages of the disease.

Postoperative staging of pancreatic cancer.

Pancreatic cancer is a devastating disease with a 5-year survival rate of 3%-5%. The mortality of pancreatic cancer is almost identical with its incidence. The vast majority are pancreatic ductal adenocarcinomas. It is typically a tumour of the elderly. The main risk factor is smoking. Clinical and histopathological studies have identified pancreatic cancer precursor lesions. These include pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasia (IPMN) and mucinous cystic neoplasm (MCN). To improve patient prognosis, surgical interventions have become more aggressive, including pancreaticoduodenectomy and more or less radical lymphadenectomy. Following surgery, it is the surgical pathologist who provides valuable information regarding the exact tumour localization, histological tumour type, grading, completeness of resection, nodal status and the presence of precursor lesions. Although many tissue-based prognostic biomarkers have been characterized and can be studied by immunohistochemistry or molecular biological techniques, their impact on patient management and treatment is still limited. More recently proteomic profiling has raised hopes for early cancer detection, thereby improving the prognosis of pancreatic cancer patients.

Expression of carbonic anhydrase 9 at the invasion front of gastric cancers.

BACKGROUND: Carbonic anhydrase IX (MN/Ca9) catalyses the reversible metabolism of carbon dioxide to carbonic acid and has also been linked to malignant transformation and hypoxia in various cancers. AIMS: To assess the expression and biological role of Ca9 in gastric cancer. METHODS: Using gastric cancer cell lines and tissues, we studied expression of Ca9 by western blot analysis, immunohistochemistry, and polymerase chain reaction. Biological changes after Ca9 transfection and after treatment with 5'-azadeoxycytidine were also analysed in cancer cell lines. RESULTS: Non-cancerous tissues strongly expressed Ca9 with membranous localisation. In contrast, Ca9 expression was frequently lost in gastric cancers (p<0.001). However, gastric cancers that retained Ca9 expression in cancer cells exhibited a shorter postoperative survival (p = 0.028). In vitro analysis revealed that loss of Ca9 expression in gastric cancer cell lines was restored after treatment with 5'-azadeoxycytidine and was associated with increased invasion (p<0.01). Moreover, AGS cells transfected with Ca9 exhibited significantly increased cell proliferation (p<0.05). CONCLUSIONS: A subgroup of gastric cancers retain Ca9 expression in cancer cells at the invasion front. While loss of Ca9 expression is regulated in part by methylation, re-expression of Ca9 is associated with increased invasion, supporting the hypothesis that increased Ca9 expression may contribute to invasion and thus advanced disease and tumour progression in a subset of gastric cancers.

Expression of carbonic anhydrase IX in human pancreatic cancer.

BACKGROUND: Carbonic anhydrase IX has been linked to cancer development and progression. AIM: To analyse carbonic anhydrase IX expression and anhydrase inhibition in pancreatic cancer and to correlate these findings with p53 expression and microvessel density. MATERIALS AND METHODS: Seventy-seven pancreatic cancers were examined (43 males, 34 females; mean age, 64 years). The anti-carbonic anhydrase IX M75 antibody was used for immunohistochemistry and Western blot analysis. Microvessels were visualized using the anti-CD34 antibody, and p53 expression in cancer cells was assessed with a specific anti-p53 antibody. Quantitative polymerase chain reaction was performed in order to assess carbonic anhydrase IX mRNA levels in the pancreas. Furthermore, pancreatic cancer cell lines were treated with acetazolamide, a carbonic anhydrase inhibitor. RESULTS: In the normal pancreas, carbonic anhydrase IX immunoreactivity was observed at the basolateral membrane of ductal cells in 24 cases (31%). Carbonic anhydrase IX expression was found at the membrane and in the cytoplasm of pancreatic cancer cells in 16 pancreatic cancers (21%). Carbonic anhydrase IX expression was independent of the localization, stage, size, metastases and differentiation of the tumour. p53 expression was significantly more frequent in poorly differentiated cancers (P=0.0323); however, p53 expression and microvessel density were independent of carbonic anhydrase IX expression. Overall, carbonic anhydrase IX expression was not altered in pancreatic cancers vs. adjacent normal pancreatic tissue as assessed by Western blot and quantitative polymerase chain reaction analysis. However, incubation of pancreatic cancer cell lines with acetazolamide led to a significant inhibition of cell proliferation in AsPC-1 and PANC-1 pancreatic cancer cells. CONCLUSION: Carbonic anhydrase IX expression is observed in both ductal epithelial and cancer cells of the pancreas. Although the expression of carbonic anhydrase IX in pancreatic cancer is not associated with angiogenesis or advanced disease, it may well be a target for carbo-anhydrase inhibitors in a subset of pancreatic cancers.

Dual role of serum soluble E-cadherin as a biological marker of metastatic development in gastric cancer.

BACKGROUND: Soluble E-cadherin serum levels as a potential biological marker for gastric cancer were analysed with special consideration to clinical and pathological features. METHODS: Seventy-one healthy control subjects and 166 patients with gastric cancer were enrolled. Gastric cancer patients were classified into intestinal-type (51%) and diffuse-type (49%), according to Laurén. Soluble E-cadherin serum levels were measured with enzyme-linked immunosorbent assay. RESULTS: The mean logarithmic concentrations of soluble E-cadherin in gastric cancer patients were significantly higher than those of control subjects, with an average of 4.03 (+/- 0.32) versus 3.86 (+/- 0.24), respectively (P < 0.0001). The concentration of soluble E-cadherin was significantly higher in the intestinal-type group than in the diffuse-type group, with an average of 4.07 +/- 0.3 versus 3.98 +/- 0.34, respectively (P = 0.0494). In the intestinal-type group, concentrations of soluble E-cadherin were significantly higher in more advanced stages (stages III-IV) than in earlier stages (stages I-II), with an average of 4.13 +/- 0.29 versus 3.96 +/- 0.31, respectively (P = 0.0234). In the diffuse-type group, concentrations of soluble E-cadherin were significantly higher in localized than in metastatic gastric cancer, with an average soluble E-cadherin concentation of 4.15 +/- 0.3 versus 3.95 +/- 0.32, respectively (P = 0.0139). CONCLUSION: Serum soluble E-cadherin concentrations exhibit a completely different pattern in intestinal-type and diffuse-typegastric cancer. Serum levels are increased in intestinal-type gastric cancer, especially in advanced stages, whereas in diffuse-type gastric cancer E-cadherin levels are decreased in advanced, metastasized cancer.We conclude that soluble E-cadherin concentrations should be interpreted along with Laurén classification and thus might serve as a biological marker in intestinal-type gastric cancer.

Absence of cyclin D2 expression is associated with promoter hypermethylation in gastric cancer.

Expression of cyclin D2 is absent in 30-70% of gastric cancers. We investigated the role of promoter hypermethylation in the transcriptional silencing of cyclin D2 in five gastric cell lines and 47 primary gastric carcinomas. CpG island methylation status of the cyclin D2 gene was studied by methylation-specific polymerase chain reaction and bisulphite sequencing. RNA and protein expression was analysed by reverse transcription-PCR and Western blot, respectively. Dense methylation of cyclin D2 was detected in three cell lines (KATOIII, AGS and NCI-N87), which also lacked cyclin D2 mRNA and protein expression. Bisulphite DNA sequencing revealed that loss of cyclin D2 expression was closely associated with the density of methylation in the promoter region. Treatment with DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the cyclin D2 expression level in methylated gastric cells. Among the 47 primary gastric cancers, cyclin D2 hypermethylation was detected in 23 (48.9%) cases. None of the 23 normal gastric biopsies from noncancer patients showed hypermethylation. Hypermethylation was associated with loss of mRNA (P&<0.001) and protein (P=0.006) expressions. Our study showed that cyclin D2 hypermethylation is associated with loss of cyclin D2 expression in a subset of gastric cancers, which may suggest an alternative gastric carcinogenesis pathway in the absence of cyclin D2 expression.

Expression of trefoil peptides (TFF1, TFF2, and TFF3) in gastric carcinomas, intestinal metaplasia, and non-neoplastic gastric tissues.

Trefoil factor family (TFF) domain peptides consist of three members that play a role in intestinal mucosal defence and repair, and in tumourigenesis. The role of the three TFF members in the gastric carcinogenesis cascade remains poorly defined. This study examined seven gastric cell lines, 50 gastric cancers and their adjacent non-cancer tissues, and tissues from 40 non-cancer patients, in order to elucidate the chronology of TFF expression in various stages of gastric carcinogenesis. TFF expression was determined by RT-PCR, immunohistochemistry, and western blot. Aberrant expression of TFF1, TFF2, and TFF3 was frequently detected in gastric cell lines. Specifically, TFF1 was detected in all non-cancer patients, but was detected in only 50% of gastric cancer and 66% of adjacent normal tissues. TFF2 expression was demonstrated in 87.5% of non-cancer patients, 34% of gastric carcinomas, and 58% of adjacent non-cancer tissues. There was a significant correlation between TFF1 and TFF2 expression in gastric cancer and adjacent non-cancer tissues (p<0.001). By contrast, TFF3 was detected in 25% of non-cancer patients and showed a predilection for areas with intestinal metaplasia (p=0.005). Sixty-two per cent of gastric cancers and 24% of neighbouring non-cancer tissues showed TFF3 expression. Immunoreactivity against TFF3 was demonstrated in goblet cells of intestinal metaplasia and within the cytoplasm and nuclei of tumour cells. Progressive loss of TFF1 and TFF2, together with the induction of TFF3, is likely to be involved in the early stage of the multi-step gastric carcinogenesis pathway.

Increased expression of survivin in gastric cancer patients and in first degree relatives.

Survivin was recently described as an apoptosis inhibitor. Its pathogenic role in gastric cancer is largely unknown. Expression of survivin in gastric cancer and non-cancer first-degree relatives, and its association with apoptosis and cyclo-oxygenase-2 expression was investigated. Fifty gastric cancer, 30 non-cancer first-degree relatives, 20 normal controls and five gastric cancer cell lines were studied. Survivin and cyclo-oxygenase-2 were evaluated by reverse transcriptase-polymerase chain reaction, immunohistochemistry and Western blot. Survivin expression was absent from normal gastric mucosa. All five cancer cell lines and 34 out of 50 (68%) human gastric cancer tissues expressed survivin mRNA. Survivin expression was less frequent (22%; P<0.001) in adjacent non-tumour gastric tissues. Immunohistochemistry and Western blot obtained similar findings. Gastric cancers with survivin expression displayed significantly reduced apoptosis (P=0.02), and associated with cyclo-oxygenase-2 overexpression at both mRNA (P=0.001) and protein levels (P=0.041). Moreover, survivin mRNA was detected in the gastric mucosa of eight (27%) non-cancer relatives. Expression in non-cancer patients showed positive correlation with H. pylori infection (P=0.004). This demonstrates the frequent expression of survivin in gastric cancer and in first-degree relatives. Co-expression of survivin and cyclo-oxygenase-2 may suggest multiple pathways contributing to the inhibition of apoptosis in gastric cancer.

Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1.

PTEN is a candidate tumour suppressor gene and frequently mutated in multiple cancers, however, not in pancreatic cancer. Recently, it has been demonstrated that PTEN expression is regulated by TGF-beta1. Using TGF-beta1 transgenic mice (n=7) and wildtype littermates (n=6), as well as pancreatic tissues obtained from organ donors (n=10) and patients with pancreatic cancer (n=10), we assessed the expression of PTEN by means of immunohistochemistry and semiquantitative PCR analysis. In addition, PANC-1 cells were treated with TGF-beta1 in vitro and the levels of PTEN mRNA were determined in these cells. In human pancreatic cancers PTEN mRNA levels were significantly decreased (P<0.05). In addition, in the pancreas of TGF-beta1 transgenic mice the expression of PTEN was significantly reduced (P<0.01), as compared to wildtype littermates and incubation of PANC-1 cells with TGF-beta1 decreased PTEN mRNA levels after 24 h. Inasmuch as TGF-beta1 decreases PTEN expression in human pancreatic cancer cells and human pancreatic cancers overexpress TGF-beta1, the reduced expression of PTEN in pancreatic cancer may be mediated by TGF-beta1 overexpression. Thus, although PTEN is not mutated in pancreatic cancers, the reduction of its expression may give pancreatic cancer cells an additional growth advantage.