Prognostic Roles Of Imprinted Gene SLC22A18 In Breast Cancer

Background SLC22A18, also known as/IMPT1/BWR1A/TSSC5, is located in the region of human chromosome 11p15.5. SLC22A18 encodes an efflux transporter-like protein with 10 transmembrane domains, whose regulation may affect drug sensitivity, cellular metabolism and growth. Human chromosome 11 p15.5 is of interest because it is frequently lost in a wide variety of tumors, including Wilms'tumor, lung cancer, hepatocarcinoma cells and breast cancer, suggesting that one or more tumor suppressor gene map to this region. The aims of this study were to evaluate the relationship of SLC22A18 expression with clinicopathologic features and investigate the prognostic value of SLC22A18 expression in breast cancer after surgery.Part 1 Differential SLC22A18 expression and invasion abilities in 2 breast cancer cell lines MDA-MB-231 and MCF-7Purpose:To investigate the differential SLC22A18 expression in 2 breast cancer cell lines MDA-MB-231 and MCF-7 and to evaluate their invasion abilities.Methods:Real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR) and western blot was applied on 2 breast cancer cell lines. Transwell was employed to evaluate the differential invasion abilities of 2 breast cancer cell lines.Results:Distinctive expression of SLC22A18 was observed in 2 breast cancer cell lines MDA-MB-231 and MCF-7. The difference was significant. Lower expression of SLC22A18 was detected in high metastasis breast cancer cell line MDA-MB-231 while higher expression of SLC22A18 was detected in low metastasis breast cancer cell line MCF-7. Transwell showed that comparing with MCF-7, breast cancer cell lines MDA-MB-231 had a much more cells that across the matrigel membrane. The distinction was notable. P=0.004.Conclusions:High metastasis breast cancer cell line MDA-MB-231 had strong invasion ability and low level of SLC22A18 expression. Imprinted gene SLC22A18 might be a tumor suppressor gene that effect metastasis of breast cancer.Part 2 Up-regulation of SLC22A18 expression on breast cancer cell linesPurpose:To up-regulate the expression of SLC22A18 on breast caner cell lines (U-231, U-7), and to identify the over-expression of SLC22A18. Compare the varied invasion abilities of up-regulation cell lines U-231,U-7 and breast cancer cell lines MDA-MB-231.MCF-7.Methods:U-231, U-7 was provided by Department of Forensic Medicine, Shanghai Medical College, Fudan University. Western Blot was applied to identify the expression of SLC22A18 protein in U-231,U-7. Transwell was employed to evaluate the differential invasion abilities of up-regulation cell lines and breast cancer cell lines.Results:Compared with MDA-MB-231, higher expression of SLC22A18 protein was detected in U-231. The difference is significant. The result remained the same when comparing MCF-7 with U-7. Transwell showed that up-regulation cell lines had lower cells that across the matrigel membrane. The distinctive was notable.Conclusions:Up-regulation cell lines were successfully constructed. Over expression of SLC22A18 may reduce the invasion abilities of breast cancer cells. SLC22A18,serve as tumor suppressor gene, may restrain the growth of tumor and weakened the invasion abilities of caner cells.Part 3 Expression of SLC22A18 in breast tissues and its relationship with clinicopathologic parameters and clinical outcomesPurpose:To detect the expression of SLC22A18 in benign breast tissues and breast cancer tissues. To analyze the correlation between SLC22A18 expression and clinicopathologic parameters in those breast cancer patients.Methods:From Jan.2004-Mar.2008,156 cases with breast cancer and 38 cases with benign breast diseases were randomly retrieved from a prospectively collected database of a total 574 cases that treated at the general surgery department, Zhongshan hospital, Fudan University, Shanghai, China. Immunohistochemistry was used to evaluate the expression of SLC22A18 protein in those breast tissues. Quantification of immunostaining was performed through digital image analysis. Statistical comparisons were done by SPSS 17.0 software to evaluate the relationship of SLC22A18 expression with clinicopathologic parameters and clinical outcomes.Results:1) We found that expression of SLC22A18 was down regulated in breast cancer and one way ANOVA analysis revealed expression of SLC22A18 was significantly higher in benign than in malignant tumors.2) The patients'age did not contribute to any significant difference in SLC22A18 expression levels.3) Breast tumor size reversely correlated with SLC22A18 expression levels. The larger size of breast cancer is, the lower expression of SLC22A18 appears.4) The expression of SLC22A18 is associated with extensive lymphovascular invasion. Lower expression was viewed in ones with extensive lymphovascular invasion than ones without extensive lymphovascular invasion.5) The expression level of SLC22A18 decreased gradually with the increasing of metastatic lymph nodes. Higher expression of SLC22A18 showed in lymph nodes positive groups than that in lymph node negative groups. However the expression level showed no significant relationship with numbers of metastatic lymph nodes.6) Estrogen receptor, progesterone receptor and HER-2 status contributed nothing to any significant difference in SLC22A18expression levels.7) The grade of tumor and the type of tumor was not related to SLC22A18 expression level.8) The expression level of SLC22A18 decreased gradually with the increasing of TNM staging.Conclusions:The expression of SLC22A18 in benign tissue was significant higher than that in malignant tumor tissue. The more advanced malignancy of breast cancer, the lower expression of SLC22A18. The trend appeared clearer as the emerging of metastatic events.Part 4 Prognostic roles of imprinted gene SLC22A18 in breast cancerPurpose:To investigate the prognostic value of SLC22A18 expression in breast cancer after surgery.Methods:We followed up all breast cancer patients through phone calling and clinical visits. We recorded the first time when recurrence or metastases occurred. The relapse-free interval (RFI) of the patients was calculated. Survival analysis was done using the Kaplan-Meier method and the Cox proportional hazards model to estimate variable with a significant independent prognostic role by means of a backward stepwise elimination procedure. P<0.05(two-sided) was judged statistically significant.Results:Kaplan-Meier analysis identified SLC22A18 expression was associated with relapse-free survival (RFS) of breast cancer. Higher expression SLC22A18 group had longer cum survival compared to the group with low expression. The difference was significant (p=0.003, log-rank test). Cox's regression analysis identified tumor size, lymph nodes metastasis, nuclear stage, extensive lymphovascular invasion, SLC22A18 expression as prognostic factor for RFS. Nuclear stage and SLC22A18 expression were the most meaning histopathologic parameter in predicting tumor recurrence. Comparing with the group showing higher expression of SLC22A18, group with lower expression had more chance to relapse. The HR is 2.624 (p=0.035).Conclusions:Less risk of relapse and metastasis could be observed in breast cancer patients with higher expression level of SLC22A18. The result find SLC22A18, a probably tumor suppressor genes, participate the development of breast cancer. Testing expression of SLC22A18 could be helpful for diagnosis and prognosis in breast cancer.