Ⅰ. The Function And Mechanism Of DAB2IP Suppressing Colorectal Cancer Metastasis Ⅱ.The Pattern And Prognosis Of Gastric Cancer Metastasis In The Mesogastrium

Objective:To dentify the biological roles of DAB2IP regulating EMT and CSC features in human colorectal cancer.Methods:We constructed stable DAB2IP-overexperssing, DAB2IP-knockdown CRC cell lines and control cells. EMT related proteins and relative transcript factors were determined by Westernblot and Real Time PCR. Meanwhile, CSC markers and related transcript factors were analysed. We further evaluted whether DAB2IP could affect anchorqge-independent growth by clone formation in soft-agar. Sphere formation assay was used to assess the number and size of sphere formed in the stable cell lines. We further compared the sensitivity to both oxaliplatin and 5-Fu in these stable cell lines though cell apoptosis assay. Limiting dilution assay will be used to compare the tumorigenicity in vivo. The EMT markers and/or CSC marker CD44 will be analysed in xenograft tumor of CRC cells, colonic tissues of DAB2IP knockout mice and pairs of clinic CRC including adjacent normal mucosa, tumor in situ and liver metastasis lesion.31 fresh colorectal cancer tissues were analyzedby the rate of CD 133+cells through flow cytometry. Finally, we compared the capacity of liver metastasis between DAB2IP-knockdown stable cells and their control stable cells in liver metastasis model.Results:Overexpression of DAB2IP can suppress the expression of EMT and CSC related proteins and genes in human colorectal cancer cells (SW480), in contrast, knocking down the endogenous expression of DAB2IP in HT29 and HCT116 cells can increase these proteins and genes. DAB2IP significantly suppress both the number and size of clones in soft-agar and spheres in CSC culture medium. In cell apoptosis assay, our data showed that reduced DAB2IP expression in CRC cells lower their sensitivity to chemotherapy drugs oxaliplatin and 5-Fu. Besides, the results of limiting dilution assay indicate that loss of DAB2IP facilitates the capacity of tumorigenicity in vivo. In xenograft tumor from HT29 and HCT116, the absence of DAB2IP promotes the E-cadherin expression. In DAB2IP knockout mice, colonic tissues showed lower expression of E-cadherin and increased expression of mesenchymal markers snail, slug and CSC marker CD44. DAB2IP and E-cadherin are reduced gradually in the adjacent normal tissue, primary tumor and liver metastasis lesion from the same patient. But, expression of snail, slug and CD44 are improved in liver metastasis tissues. In the 31 fresh CRC tissues, further analysis using flow cytometry shows that the decreased expression of DAB2IP can enrich more CD133+ cells in CRC patients. Last, we found that loss of DAB2IP in CRC promoter cancer liver metastasis in liver metastasis model.Conclusions:DAB2IP could suppress EMT and CSC properties of colorectal cancer both in vitro and vivo. Moreover, absence of DAB2IP could promote CRC metastasis.Objective:To explore the possible mechanisminvolved by DAB2IP in regulating EMT and CSC properties in colorectal cancer.Methods:We collected nucleus proteins in stable DAB2IP overexpression (SW480) and knock-down (HT29, HCT116) cells and examined the p65 protein expression by Western blot. The NF-κB transcriptional activity was detected by dual-luciferase reporter assay. To confirm whether the inhibitory effect of the DAB2IP in regulating EMT and CSC properties could be reversed by activatingNF-κB signal pathway, the NF-kB transcriptional activity, EMT marker expression and the capacity of sphere formation were analyzed after transfectingsi-IκB-αor si-p65 by small interfering RNAs. Moreover, we tested the expression of p65 and IκB-α in the xenograft tumor and in theclinic colorectal cancer tissues.Results:Compared to these control cells, p65 expression was decreased in SW480-D2 cells and increased in HT29-KD5 and HCT116-KD3 cells. DAB2IP inhibited the NF-kB transcriptional activity in these CRC cells. In SW480-D2 cells, si-IxB-α could reverse the inhibitory effect of the NF-kB transcriptional activity by DAB2IP over-expression. Similary, si-p65 could reverse the activation effect of the NF-kB transcriptional activity by knocking down DAB2IP in HT29-KD6 and HCT116-KD3 cells. Besides, We found that the decreased expression of DAB2IP promoted translocating p65 to nucleus and IkB-α out of nucleus in xenograft tumor tissue.The similar results were observed in DAB2IP knockout mice and clinic colorectal cancer tissues.Conclusions:DAB2IP regulates EMT and CSC features through NF-kB pathway in CRC.Objective:To determine the relationship between DAB2IP expression and prognosis in colorectal cancer patients.Methods:We analysed the expression of DAB2IP in the CRC tumor, adjacent normal mucosa, the tumor invasive front and the center region of the same slices by immunohistochemistery (IHC). The DAB2IP expression pattern of 162 CRC tissues, 100 adjacent normal tissues and 62 metastasis lesions tissues were analysed. Moreover, we further detected the correlation between DAB2IP expression and clinic-pathological factors in the 162 CRC tissues. Kaplan-Meier survival analysis was used to evaluate the prognostic effects of DAB2IP on CRC patients’survival.Results:We found that DAB2IP was distinctly down-expression in the CRC tissue in comparison with adjacent normal mucosa tissue in the same slices. The expression of DAB2IP in the tumor invasive front was significant lower than in the center region. In cohort of CRC tissues, our data showed that in adjacent normal mucosa tissues only 8%(8/100) cases exhibited low or no expression of DAB2IP, while 92% (92/100) cases exhibited medium or high expression. In CRC tissues, 62.3%(101/162) manifested a low or no expression of DAB2IP, however, up to 80.6%(50/62) cases showed the absence expression of DAB2IP in metastasis lesions. Relationship analysis between DAB2IP and clinic-pathological factors in the 162 CRC tissues indicated that the loss of DAB2IP strongly correlated with metastasis (P=0.034) and differentiation (P=0.004). Moreover, patients with low DAB2IP expression showed significant lower 5-year survival rates than those with high DAB2IP expression (P=0.001) by Kaplan-Meier survival analysis.Conclusions:DAB2IP is an important prediction factor for CRC patient prognosis. Decreased DAB2IP expression facilitates tumor progress in CRC patients.Objectives:To evaluate the incidence and pattern of Metastasis V in gastric cancer.Methods:We detected metastatic cancer cells in the mesogastrium by H&E and immunohistochemistry (IHC) in large cross sectional tissue. Besides, we analysed the relationship between clinicopathological factors of patientsandMetastasis V.Results:Metastasis V was detected in 9 of 74 (12.2%) patients with gastric cancer, and 1 of 40 (2.5%) patients with early gastric cancer,8 of 34 (24%) patients with advanced gastric cancer. The mean distance of Metastasis V from gastric wall was approximately 2.6 cm. The incidence rate of Metastasis Vwas positively correlated with tumor invasion depth (P=0.001) and the number of positive lymph node metastasis (P=0.001). But no significant correlation was to be found with gender, age, tumor size and differenciation.Conclusions:Metastasis V can be detected in the mesogastrium of both early and advanced gastric cancer patients by using whole-sectional analysis. The incidence of Metastasis V was closely related to the depth of the primary tumor invasion and the number of positive lymph node metastasis.Objectives:To dentify whether Metastasis V could affect the prognosis of gastric cancer patients.Methods:We followed up with the patients and compared the 3-year survival rate of patients between Metastasis V positive and negative using Kaplan-Meier survival curves analysis.Univariate analysis was used to evaluate the clinic-pathological factors affecting prognosis. Multivariate analysis was also used to dentify whether Metastasis V could be an independent factor evaluating prognosis of gastric cancer patients.Results:Based on our follow-up datas, we found that the prognosis of Metastasis V-positive patients was significant (P=0.006) worse than those of MetastasisV-negative patients. In addition, the prognosis of the patients with Metastasis V-positive was significant poorer than those with Metastasis V-negative in the T3 group (P=0.004) and in clinic stage II (P=0.0005). According to a univariate analysis, the size of tumor (P=0.02), Laurdn classification (P=0.01) and Metastasis V (P=0.002) were significant correlated with patient survival. In a multivariate analysis, Metastasis V (P=0.037) was found to be an independent factor for predicting prognosis of patients with gastric cancer.Conclusions:Metastasis V in gastric cancer could affect patients’prognosis and it could be an independent factor for predicting prognosis of patients with gastric cancer.