The Role Of Wnt Antagonist SFRP1 In Gastrointestinal Tumors

The role of Wnt antagonist SFRP1 in gastrointestinal tumorsPrefaceWnt signaling pathway plays important roles in proliferation, differentiation, and apoptosis in adult tissues. Thus aberrant activation of Wnt pathway may induce tumorigenesis. In recent years, upstreams in Wnt pathway involving carcinogenesis have been observed. Overexpression of members of Wnt family, such as Wnt1, Wnt2 and Wnt3a, has been found in several human cancers. In addition, aberrant activation of Wnt pathway by downregulation of Wnt antagonists can also induce tumorigenesis. For example, downregulation of Dkk family has been found in several cancers. Recently, expression loss of SFRP1 has been reported in human cancers.Gastrointestinal cancer is one of the most common human cancers. It is very important to study the pathogenesis and methods for early detection and treatment. Lots of studies have shown that the pathogenesis of gastrointestinal cancer is a multiple genes-involved process, including mutations of tumor inhibitor genes and activation of pro-oncogenes. Additionally, the role of epigenetic change in gastrointestinal cancer has also been studied. In this study, we analyzed the expression of SFRP1 and its mechanism in gastrointestinal cancer, and studied the role of aberrant expression of SFRP1 in gastrointestinal carcinogenesis.Some studies have shown overexpression of Wnt enhances the metastasis and invasiveness of tumors. Downregulation of SFRP1 and the subsequent activation of Wnt pathway have been found in several human cancers. So the correlation of SFRP1 downregulation with tumor metastasis and invasiveness in gastrointestinal cancer is a question needed to further study. Of the factors involved in gastrointestinal tumor metastasis, overexpression or activation of MMPs is one of the important mechanisms. MMP-9 and MMP-2 in MMPs family can breakdown collagenⅣin basal membrane. Overexpression of MMP-2 or MMP-9 can enhance the metastasis and invasiveness of gastrointestinal tumor. In this study, we studied the correlation of downregulation of SFRP1 with metastasis and invasiveness of gastrointestinal tumor, and determined the mechanism by detecting the levels of MMP-2 and MMP-9.Materials and methods1. Materials52 gastric cancers, 60 colorectal cancers, and their matched adjacent tissue samples were all obtained from the second affiliated hospital, Chinese Medical University. The samples were frozen in liquid nitrogen immediately after surgery. Human gastric cancer cell lines SGC-7901, BGC-823, HGC-27 and colon cancer cell lines HT-29, SW-480 were all purchased from KUNKEN Bio-reagent Corp.,Shanghai, China. Trizol, RNA PCR kit and LightCycler DNA Master SYBR GreenⅠkit were all from Takara. Methylation detection kit was purchased from GENMED. Antibodies of SFRP1, MMP-2 and MMP-9 were purchased from SANTA CRUZ.2. MethodsHuman gastric cancer cell lines SGC-7901, BGC-823 and HGC-27 and colon cancer cell lines HT-29 and SW-480 were cultured in RPMI 1640 supplemented with 10％fetal bovine serum, penicillin (100IU/ml), and streptomycin(100μg/ml). DNA was extracted by satured NaCl method and RNA was extracted by Trizol methods. SFRP1 mRNA was detected by RT-PCR. MMP-2 mRNA and MMP-9 mRNA were measured by real time PCR. Hypermethylation of SFRP1 was detected by methylation specific PCR (MSP). MMP-2 and MMP-9 proteins were determined by Western blotting.3. Statistical analysis Methylation and expression status of SFRP1 between cancer and cancer adjacent tissues were compared byχ~2 test. Fisher's exact test was used to study the statistical association between clinical pathologic data and aberrant expression of SFRP1. MMPs mRNA between different cells were compared by student's t test. Differences were considered statistically significant when P values were less than 0.05.Result1. hypermethylation and expression of SFRP1 in gastric cancer cell linesHypermethylation of SFRP1 was found in BGC-823 and HGC-27, not in SGC-7901.Expression of SFRP1 mRNA was detected in SGC-7901, but not in BGC-823 and HGC-27. After treatment of BGC-823 and HGC-27 with the demethylating agents, 5-aza-2'-deoxycytidine, SFRP1 mRNA was re-expressed in BGC-823 and HGC-27.2. Hypermethylation and expression of SFRP1 in gastric cancers and matched cancer adjacent tissues.In 52 gastric cancers, hypermethylation of SFRP1 was detected in 23 cases. In matched cancer adjacent tissues, 8 cases were found with SFRP1 hypermethylation. The hypermethylation rate in gastric cancers is significant higher than that in matched cancer adjacent tissues(P＜0.01). Expression of SFRP1 mRNA was not found in 16 and 6 cases in gastric cancers and matched cancer adjacent tissues respectively. The difference is also significant(P＜0.01). Loss of SFRP1 is correlated with SFRP1 hypermethylation(P＜0.001).3. Hypermethylation and expression of SFRP1 in colon cancer cell linesHypermethylation of SFRP1 was found in SW-480, not in HT-29.Expression of SFRP1 mRNA was detected in HT-29, but not in SW-480. After treatment of SW-480 with the demethylating agents, 5-aza-2'-deoxycytidine, SFRP1 mRNA was re-expressed in SW-480.4. Hypermethylation and expression of SFRP1 in colorectal cancers and matched cancer adjacent tissues.In 60 colorectal cancers, hypermethylation of SFRP1 was detected in 38 cases. In matched cancer adjacent tissues, 11 cases were found with SFRP1 hypermethylation. The hypermethylation rate in colorectal cancers is significant higher than that in matched cancer adjacent tissues (P＜0.01). Expression of SFRP1 mRNA was not found in 35 and 8 cases in colorectal cancers and matched cancer adjacent tissues respectively. The difference is also significant (P＜0.01). Loss of SFRP1 is correlated with SFRP1 hypermethylation (P＜0.001).5. Correlation analysis of SFRP1 expression with clinical pathologic data in gastric cancers and colorectal cancers.In gastric cancers, SFRP1 expression was correlated significantly with tumor stage (P=0.01) and lymph node status (P=0.04), but not with patient sex, age, tumor size and histological type. In colorectal cancers, SFRP1 expression was also correlated significantly with tumor stage (P=0.01) and lymph node status (P=0.01), but not with patient sex, age and tumor size.6. Expressions of MMP-2 mRNA and MMP-9 mRNA in gastric cancer cell lines and colon cancer cell linesIn gastric cell lines, SGC-7901 expressed low levels of MMP-2 mRNA and MMP-9 mRNA, while BGC-823 expressed high levels of MMP-2 mRNA and MMP-9 mRNA. The difference is significant (P＜0.01). In colon cancer cell liness, HT-29 expressed low levels of MMP-2 mRNA and MMP-9 mRNA, while SW-480 expressed high levels of MMP-2 mRNA and MMP-9 mRNA. The difference is also significant (P＜0.01). 7. Expressions of MMP-2 and MMP-9 proteins in gastric cancer cell lines and colon cancer cell linesIn gastric cell lines, SGC-7901 expressed low levels of MMP-2 and MMP-9 proteins, while BGC-823 expressed high levels of MMP-2 and MMP-9 proteins. In colon cancer cell liness, HT-29 expressed low levels of MMP-2 and MMP-9 proteins, while SW-480 expressed high levels of MMP-2 and MMP-9 proteins.8. Treatment of BGC-823 and SW-480 with the demethylating agents, 5-aza-2'-deoxycytidine.After treatment of BGC-823 and SW-480 with the demethylating agents, 5-aza-2'-deoxycytidine, expressions of MMP-2 mRNA and MMP-9 mRNA decreased significantly (P＜0.01).Conclusion(1) SFRP1 expression is lost in some gastric cancers. SFRP1 expression lost is caused mainly by SFRP1 gene hypermethylation. The results indicate that SFRP1 expression loss is involved in the pathogenesis of some gastric cancers.(2) SFRP1 expression is lost in some colorectal cancers. SFRP1 expression lost is caused mainly by SFRP1 gene hypermethylation. The results indicate that SFRP1 expression loss is also involved in the pathogenesis of some colorectal cancers.(3) SFRP1 expression loss enhances the metastasis and invasiveness of gastric and colorectal cancers.(4) SFRP1 expression loss upregulates the expressions of MMP-2 and MMP-9, which may be the mechanism by which SFRP1 expression loss enhances the metastasis and invasiveness of gastric and colorectal cancers.