Guang-Yun Yang

Integrin alphavbeta6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver

Integrin alphavbeta6 (αvβ6) is correlated with colon cancer progression. To detect the effects of αvβ6 on liver metastasis, the specificity of αvβ6 against the monoclonal antibody (mAb) 2G2 was examined by immunoprecipitation. Integrin αvβ6‐immunoreactivity (IR) in liver metastasis tissues (63 cases) and colon carcinoma (358 cases) were examined. These results showed that αvβ6 was specifically recognized by the mAb 2G2, and that rates of αvβ6 positivity in liver metastatic tissues (71.4%, 45/63) were higher than that for primary colon cancer (34.0%, 122/358) (P < 0.01). Patients who were αvβ6‐positive had higher liver metastasis rates (17%, 21/122) than those who were αvβ6‐negative (only 3%, 7/236) (P < 0.01). To examine the underlying mechanisms associated with αvβ6 regulating colonic metastasis in the liver, experimental liver metastasis (intrasplenic injection of HT29 transfectants) and liver colonization assays (direct injection of WiDr transfectants into the liver) in nude mice were performed; these demonstrated that αvβ6 contributed to the promotion of the metastatic potential and the survival of cancer cells in the liver. Matrix metalloproteinase‐9 (MMP‐9) levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP‐9 activity assay system and gelatin zymography assay, and showed that suppression of αvβ6‐IR inhibited MMP‐9 activity and secretion. Transwell migration assay in vitro also showed that αvβ6 promoted migration on fibronectin for HT29/WiDr mock compared with HT29/WiDr antisense β6 transfects (P < 0.01). We concluded that αvβ6 may mediate the potential for colon cancer cells to colonize in and metastasize to the liver. The mechanisms that αvβ6 may be involved in include the promotion of MMP‐9 secretion, the enhancement of migration on fibronectin, and the survival of cancer cells in the liver. (Cancer Sci 2008; 99: 879–887)

Vascular endothelial growth factor (VEGF) enhances gastric carcinoma invasiveness via integrin alpha(v)beta6

Integrins play an important role in tumor metastasis induced by vascular endothelial growth factor (VEGF). However, in the case of gastric cancer, the precise role of VEGF in regulating integrin alphavbeta6 is unclear. In this study, we found that most of the alphavbeta6 integrin-positive gastric cancer tissues were also VEGF-positive. Furthermore, when gastric carcinoma cells were exposed to VEGF, expression of alphavbeta6 integrin was up-regulated and the extracellular signal-related kinase (ERK) pathway was activated. When integrin alphavbeta6 was blocked either with beta6 siRNA or anti-alphavbeta6 antibody, the migration of tumor cells normally induced by VEGF, as well as the activation of ERK, were markedly inhibited. Blocking the ERK signaling pathway significantly inhibited cell mobility. Taken together, the data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin alphavbeta6 expression and activation of ERK.

Suppression of integrin αυβ6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway

Integrin αυβ6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of αυβ6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down‐regulate αυβ6 expression in protein and mRNA levels. Supression of integrin αυβ6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro‐MMP‐9 and pro‐MMP‐2 in tumor conditioned medium, and more important, inhibits MAPK‐dependent [3H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin αυβ6 inhibits extracellular matrix degradation through the MAPK pathway.

Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression

AIM To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices. METHODS An immunohistochemical study of integrin αvβ6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors. RESULTS High immunoreactivity for αvβ6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvβ6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvβ6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvβ6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvβ6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvβ6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvβ6 expression and promoted MMP-9 secretion compared with low density. CONCLUSION Integrin αvβ6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανβ6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.

Integrin αvβ6 and matrix metalloproteinase 9 correlate with survival in gastric cancer.

AIM To investigate the expression of integrin αvβ6 and matrix metalloproteinase 9 (MMP-9), their association with prognostic factors and to assess their predictive role in gastric cancer patients. METHODS Immunohistochemistry was used to determine the expressions of integrin αvβ6 and MMP-9 in 126 specimens from patients with primary gastric carcinoma. Associations between immunohistochemical staining and various clinic pathologic variables of tissue specimens were evaluated by the χ(2) test and Fishers exact test. Expression correlation of αvβ6 and MMP-9 was assessed using bivariate correlation analysis. The patients were followed-up every 3 mo in the first two years and at least every 6 mo afterwards, with a median follow-up of 56 mo (ranging from 2 mo to 94 mo). Four different combinations of αvβ6 and MMP-9 levels (that is, both markers positive, both markers negative, αvβ6 positive with MMP-9 negative, and αvβ6 negative with MMP-9 positive) were evaluated for their relative effect on survival. The difference in survival curves was evaluated with a log-rank test. Survival analysis was conducted using the Kaplan-Meier survival and Cox proportional hazards model analysis. RESULTS The expressions of integrin αvβ6 and MMP-9 were investigated in 126 cases, among which 34.92% were positive for αvβ6 expression, and 42.06% for MMP-9 expression. The expression of αvβ6 was associated with Lauren type, differentiation, N stage, and TNM stage (the P values were 0.006, 0.038, 0.016, and 0.002, respectively). While MMP-9 expression was associated with differentiation, T stage, N stage, and TNM stage (the P values were 0.039, 0.014, 0.033, and 0.008, respectively). The positive correlation between αvβ6 and MMP-9 in gastric cancer was confirmed by a correlation analysis. The Kaplan-Meier survival analysis showed that patients with expression of αvβ6 or MMP-9 alone died earlier than those with negative expression and that patients who were both αvβ6 and MMP-9 positive had a shorter overall survival than those with the opposite pattern (both αvβ6 and MMP-9 negative) (P = 0.000). A Cox model indicated that positive expression of αvβ6 and MMP-9, diffuse Lauren type, as well as a senior grade of N stage, M stage, and TNM stage were predictors of a poor prognosis in univariate analysis. Only αvβ6 and MMP-9 retained their significance when adjustments were made for other known prognostic factors in multivariate analysis (RR = 2.632, P = 0.003 and RR = 1.813, P = 0.007). CONCLUSION The expression of αvβ6 and MMP-9 are closely correlated, and the combinational pattern of αvβ6 and MMP-9 can serve as a more effective prognostic index for gastric cancer patients.

Hepatoma-derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright

Modified method of hepatic portal choledochoplasty to treat benign strictures of hilar biliary ducts

Background and Aim:  Roux‐en‐Y hepaticojejunostomy (RYHJ) is usually used to treat benign strictures of hilar bile ducts. However, RYHJ might also induce ascending cholangitis and recurrent hepatolithiasis. The present study aims to introduce a modified hepatic portal choledochoplasty with a pedicled graft of gallbladder (HPC) to treat this disease.