Novel focal adhesion protein kindlin-2 promotes the invasion of gastric cancer cells through phosphorylation of integrin ß1 and ß3.

BACKGROUND: We have found that the expression of the novel focal adhesion protein kindlin-2 had a significant positive correlation with poor survival in gastric cancer. However, the mechanism by which kindlin-2 acts in gastric cancer warrants further evaluation. METHODS: Kindlin-2 mRNA expression in gastric cancer cell lines was measured by realtime RT-PCR under normal and hypoxic conditions. Cell proliferation, apoptosis, cell cycle, tumor adhesion, cell invasion ability, and phosphorylation of integrin ß1 and ß3 proteins were measured to assess the influence of kindlin-2 on the malignant behavior of gastric cancer cells. RESULTS: Kindlin-2 mRNA expression was highest in the distant metastasis gastric cancer cell line Hs-746T. Cell proliferation, adhesion with endothelium and collagen IV, invasion rate, and angiogenesis genes expression, as well as phosphorylation of integrin ß1 and ß3 in Hs-746T, were decreased significantly after kindlin-2 downregulation, but there was no change in apoptosis and cell cycle. CONCLUSIONS: Kindlin-2 might promote the invasion of gastric cancer cells through enhancing proliferation and adhesion by the phosphorylation of integrin ß1 and ß3.

Macrophage coculture enhanced invasion of gastric cancer cells via TGF-ß and BMP pathways.

OBJECTIVE: Transforming growth factor ß (TGF-ß) superfamily plays an important role in regulating gastric cancer progression. As previously demonstrated, tumor-associated macrophages (TAMs) promoted the invasion of gastric cancer cells in Matrigel. However, the role of TGF-ß superfamily signaling between TAMs and gastric cancer remains unclear. MATERIAL AND METHODS: Three-dimensional dynamic migration imaging system was used to detect gastric cancer invasion rate cocultured with macrophages in Matrigel before or after TGF-ß receptor 1 or bone morphogenic protein (BMP) receptor 1A and 1B inhibition; real-time RT-PCR was used to quantitatively investigate gene expression (TGF-ß1, TGF-ß2, BMP4, and BMP7, ADAM9, MMP9, TIMP3, VEGF-A, and VEGF-C). RESULTS: TGF-ß1, TGF-ß2, BMP4, and BMP7 expressions were increased significantly in macrophages grown with cancer cells as compared to macrophages grown alone. The invasion rate and invasion-related genes expressions of both AGS and Hs-746T gastric cancer cell lines were upregulated by macrophages, although the expression profile was different. Invasion rate and invasion-related genes' expressions of AGS cells cocultured with macrophages were downregulated significantly after TGF-ßR1 and BMPR1 inhibition. CONCLUSIONS: Macrophages associated with tumor might promote gastric cancer cells invasion though enhancing TGF-ß/BMPs signal pathway. Inhibiting TGF-ß/BMPs signal between TAMs and gastric cancer cells might provide a new therapeutic method of gastric cancer.

Both macrophages and hypoxia play critical role in regulating invasion of gastric cancer in vitro.

BACKGROUND: As previously demonstrated, tumor associated macrophages (TAMs) infiltration is associated with some cancers invasion and metastasis. However, the role of TAMs in the gastric cancer remains unclear. METHODS: Three- dimensional dynamic migration imaging system and real time RT-PCR were used to quantitatively investigate the effect of macrophages on the cancer cell mobility and gene expression related to cancer invasion and metastasis, including ADAM8, ADAM9, MMP9, TIMP3, VEGF-A and IL8 genes, in AGS, HGC-27, Hs-746T and NCI-N87 gastric cancer cell lines under normal or hypoxic conditions. RESULTS: Under normal conditions, the cancer cell invasion rate was increased significantly and all six gene expressions were upregulated in all four cancer cell lines by macrophages. Under hypoxia the changes in the cancer cell invasion rate induced by macrophages was negatively correlated to the TIMP3 expression. In non- metastatic cell line AGS, the increase in migration rate induced by macrophages was further elevated under hypoxia with increased ADAM8 and ADAM9 expression and decreased MMP9 and TIMP3 expressions. Under hypoxia, the induction by macrophages for IL-8 expression was increased significantly in distant metastatic cell lines NCI-N87 and HS-746T, VEGF-A was increased in HGC-27 cell line. CONCLUSIONS: Both macrophages and hypoxia play an indispensable role in regulating the invasion of gastric cancer cells in vitro; ADAMs, MMP9 and TIMP3 might be involved in TAM induced invasive power of gastric cancer cells.

Long-term results of ablation with antireflux surgery for Barrett's esophagus: a clinical and molecular biologic study.

BACKGROUND: The initial results from ablation therapy for metaplastic/dysplastic Barrett's esophagus (BE) are promising, but the results of extended follow-up evaluation are seldom reported. METHODS: Neodymium:yttrium-aluminum-garnet laser ablation and successful antireflux surgery for 18 patients with metaplastic BE primarily resulted in the total histologic eradication of BE in 15 patients (83%). After antireflux surgery, the healing of gastroesophageal reflux disease (GERD) was objectively verified in all the patients. At late follow-up evaluation, endoscopy, conventional histology, molecular oxidative stress analyses in comparison with normal control conditions (8-hydroxydeoxyguanosine [8-OHdG], superoxide dismutase [SOD], glutathione [GSH], myeloperoxydase [MP]), and immunohistochemistry (p53, and Cdx2, caudal-related homeobox gene 2, marking intestinal differentiation) of the neosquamous epithelium were performed. RESULTS: At the end of the follow-up period (range, 3-15 years; mean, 8 years), intestinal metaplasia without dysplasia was detected histologically in eight patients (44%). Six patients had macroscopic BE (mean length, 3.5 cm; range 1-10 cm). The neosquamous epithelium was histologically normal, with no underlying columnar tissue. The fundoplication was endoscopically normal in 14 patients (82%). The 8-OHdG level was higher in the neosquamous epithelium than in the control conditions in the distal esophagus (4.3 vs. 0.52; P = 0.0002) and the proximal esophagus (1.8 vs. 0.95; P = 0.006). Likewise, SOD activity was higher in the neosquamous epithelium (0.38 vs. 0.12; P = 0.0005), whereas MP activity and GSH levels remained normal. Three patients showed slight nuclear p53 expression (typical in normal inflammatory reactions), whereas Cdx2 positivity was confined to one case with recurrent intestinal metaplasia. CONCLUSIONS: The neosquamous mucosa, generated by the ablation of BE and the treatment of GERD with fundoplication, was stable during long-term follow-up evaluation in two-thirds of the patients with initial eradication. It had normal p53 expression and no Cdx2 protein expression. The oxidative stress of the neosquamous esophagus remained high, although the clinical significance of this is unclear.

Genetic association and interaction analysis of USF1 and APOA5 on lipid levels and atherosclerosis.

OBJECTIVE: USF1 is a ubiquitous transcription factor governing the expression of numerous genes of lipid and glucose metabolism. APOA5 is a well-established candidate gene regulating triglyceride (TG) levels and has been identified as a downstream target of upstream stimulatory factor. No detailed studies about the effect of APOA5 on atherosclerotic lesion formation have been conducted, nor has its potential interaction with USF1 been examined. METHODS AND RESULTS: We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with precise quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated. CONCLUSIONS: Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.

CIP2A expression and prognostic role in patients with esophageal adenocarcinoma.

CIP2A is overexpressed in many cancers, including esophageal squamous cell carcinoma. The regulation of c-MYC and CIP2A expression is characterized by a positive feedback mechanism facilitating the expression of both of them and accelerating cancer cell proliferation in gastric cancer. Increased CIP2A expression is a predictor of poor survival in some cancers. The incidence of positive CIP2A immunostaining and its association with c-MYC and its predictive value in esophageal adenocarcinoma are unknown. All esophageal adenocarcinoma patients from 1990 to 2007 with sufficient material for analysis of CIP2A and c-MYC in two university hospitals were included in the study. In addition, biopsies from Barrett's epithelium from the cancer patients and control tissue from normal esophageal mucosa adjacent to the tumor were included. CIP2A was moderately or strongly positive in 77.9 %, and c-MYC in 93.8 % of the cancer specimens. These frequencies were statistically different from the expression in normal esophageal epithelium. In addition, there was a positive correlation between CIP2A and c-MYC expression (p = 0.018). According to adjusted Cox regression survival analysis, CIP2A and c-MYC had no effect on survival. However, among patients with stage IVA-IVB cancer, there was a trend toward poor prognosis in CIP2A-positive patients. The expression of CIP2A and c-MYC was associated with each other, and their overexpression was found in most cases of esophageal adenocarcinoma. However, CIP2A and c-MYC had no effect on survival.

Vasohibin-1 expression is regulated by transforming growth factor-ß/bone morphogenic protein signaling pathway between tumor-associated macrophages and pancreatic cancer cells.

Vasohibin-1 has been detected in endothelial cells as an intrinsic angiogenesis inhibitor. Both tumor-associated macrophages (TAMs) and transforming growth factor-ß (TGF-ß)/bone morphogenic protein (BMP) signaling have been reported to promote angiogenesis in cancer. However, whether vasohibin-1 expression is regulated by TGF-ß/BMP signaling between TAMs and cancer cells remains unclear. The expression of TGF-ß1, TGF-ß2, BMP-4, and BMP-7 in TAMs and the expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and VEGF-C in two pancreatic cancer cell lines (a nonmetastatic cell line Panc-1 and a distant metastatic cell line HPAF-II) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The TGF-ß receptor 1 and BMP receptor 1 were inhibited by the inhibitor SB-431542 and LDN193189, respectively. Thereafter, vasohibin-1, VEGF-A, and VEGF-C expression was detected by real-time RT-PCR. We found that the expression of TGF-ß1, TGF-ß2, BMP-4, and BMP-7 was upregulated in TAMs cocultured with pancreatic cancer cells. Vasohibin-1, VEGF-A, and VEGF-C mRNA expression in pancreatic cancer cells was upregulated by TAMs. Vasohibin-1 expression in pancreatic cancer cells cocultured with TAMs was upregulated significantly when TGF-ß receptors or BMP receptors were inhibited, but VEGF-C expression was downregulated. Therefore, Vasohibin-1 expression is regulated by the TGF-ß/BMP signaling between TAMs and pancreatic cancer cells. These results might shed a new light on the antiangiogenesis therapy in the pancreatic cancer.

The novel focal adhesion gene kindlin-2 promotes the invasion of gastric cancer cells mediated by tumor-associated macrophages.

Kindlin-2 is a novel focal adhesion gene mediating the cell-extracellular matrix (ECM) adhesion. Tumor-associated macrophages (TAMs) play an important role in linking chronic inflammation to cancer progression. Both kindlin-2 and TAMs have been found to promote the invasion of gastric cancer cells in our previous studies. However, the correlation between kindlin-2 and TAMs remains unclear. Real-time RT-PCR was used to investigate kindlin-2 expression in the AGS, NCI and Hs-746T gastric cancer cell lines co-cultured with TAMs under normal or hypoxic conditions. IL8, IL10, IL11, IL17b, IL18, IL22 and IL24 expressions were measured by real-time RT-PCR in the gastric cancer lines with varying levels of kindlin-2 expression, as well as after downregulation of kindlin-2 mRNA expression by the siRNA method. We found that kindlin-2 was upregulated in all three gastric cancer cell lines when co-cultured with TAMs under normal conditions. Under hypoxic conditions, the induction of kindlin-2 expression induced by macrophages was significantly downregulated in the Hs-746T cell line. IL8, IL11, IL17b, IL22 and IL24 expression was significantly higher in gastric cell lines with high kindlin-2 expression. Downregulation of kindlin-2 mRNA decreased IL10, IL11, IL17b, IL22 and IL24 expression but IL8 and IL18 expression was upregulated. Therefore, the novel focal adhesion gene kindlin-2 may play an important role in promoting the invasion of gastric cancer cells mediated by TAMs through regulating interleukin expression.

Vasohibin-1 and vasohibin-2 expression in gastric cancer cells and TAMs.

Accumulating evidence suggests that TAMs contribute to tumor progression. Recently, vasohibin-1 and vasohibin-2 were detected in endothelial cells and considered as intrinsic angiogenesis inhibitors. However, it is not known whether they are also expressed in cancer cells or tumor-associated macrophages (TAMs). Realtime RT-PCR was used to investigate the vasohibin-1 and vasohibin-2 expression in four gastric cancer cell lines, including a non-metastatic cell line AGS, and metastatic cell lines HGC-27, Hs-746T and NCI-N87, co-cultured with or without TAMs. The effect of hypoxic conditions on vasohibin expression was evaluated as well, and the correlation between vasohibin-1, vasohibin-2 and VEGF-A expression under different culture conditions was analyzed. We found that both vasohibin-1 and vasohibin-2 were expressed in the four gastric cancer cell lines and in TAMs. Under normal conditions, vasohibin-1 and vasohibin-2 expressions were significantly upregulated by TAMs in all the gastric cancer cell lines. Under hypoxia, both vasohibin-1 and vasohibin-2 expressions were significantly decreased in the distant metastasis cancer cell line Hs-746T, cultured with or without TAMs (P<0.001). After induction by TAMs or hypoxia, the vasohibin-1 and vasohibin-2 expressions correlated with that of VEGF-A. In addition, TAMs, when co-cultured with the metastatic cancer cell lines, showed hypoxia-induced vasohibin-1 upregulation (P<0.05). In conclusion, both vasohibin-1 and vasohibin-2 mRNA are expressed in gastric cancer cells and in TAMs, and their expressions are altered by hypoxia.

Esophageal adenocarcinoma arising after antireflux surgery: a population-based analysis.

OBJECTIVE: Fundoplication is widely used to treat gastroesophageal reflux disease (GERD). Whether it diminishes the development of esophageal adenocarcinoma (EAC) is, however, controversial. Our aim was to define, at the national level in Finland, frequency and predisposing factors for post-fundoplication EAC. METHODS: For this population-based study from 1980 to 2006, Finland's administrative databases provided preliminary data. Analyses of EAC patient records (N = 1035) led us to include those with preceding antireflux surgery. Conservatively treated patients were not analyzed. The EAC incidence in patients with antireflux surgery was compared with that in the general population (1987-2006) by means of standardized incidence ratio (SIR). RESULTS: A total of 53 (5.1%) EAC patients had undergone antireflux surgery. Of these patients with male predominance (74%), preoperatively 41 (77%) had developed endoscopic esophagitis, 40 (75%) hiatal hernia, 24 (45%) Barrett's esophagus (BE), nine (17%) ulcer in the esophagus or gastroesophageal junction, and three (6%) stricture. Postoperatively, histologically confirmed BE was present in 42 (79%). Antireflux surgery had preceded EAC at a mean interval of 10.1 years (range 0.5-25.6 years). This interval was significantly (p=0.02) shorter in patients with long-term functioning fundoplication (n = 15; 30%) at EAC diagnosis (6.4 years, range 0.5-15.2 years) than in those (n = 22, 44%) with failure (11.2 years, range 4.0-24.3 years). Overall, the SIR for EAC after antireflux surgery (1987-2006) was 9.21. CONCLUSIONS: Intention-to-treat GERD with antireflux surgery does not prevent EAC. It often develops more than 5 years postoperatively, also in the patients with a good antireflux barrier. Only one-third of the patients had, however, a functioning fundoplication. Preoperative BE and endoscopic esophagitis may be risk factors. Prospective, long-term, randomized studies in experienced centers may reveal the definite effect of antireflux surgery on EAC development.