Expression Of CA IX In Human Breast Cancer And The Involvement In Regulation Of Migration And Invasion/Metastasis

IntroductionBreast cancer is one of the major causes of death in women. Among various prognostic indicators of breast cancer, disease recurrence and metastasis are key factors affecting the outcome of primary breast cancer therapy. Identification of sensitive and specific biomarkers for metastatic potential and their regulatory genes in breast cancer cells, therefore, is important for both early clinical detection and effective prophylactic therapy.Hypoxia, a common consequence of solid tumor growth in breast cancer and other cancers, serves to propagate a cascade of molecular pathways that include angiogenesis, glycolysis, and alterations in microenvironmental pH. Hypoxia stabilizes HIF-la, which then triggers the expression of several target genes. CA Ⅸ, as one of the downstream products of HIF-1α, is considered to be a reliable hypoxic marker. CA Ⅸ is implicated in cell adhesion as well as in acid-base balancing and intercellular communication, which plays a critical role in malignant tumor progression.Using two experimental models-respected clinical specimens of human breast cancer and human breast cancer cell lines-this study investigated the expression of CA IX in breast cancer and its involvement in the regulation of migration and invasion/metastasis, in an effort to definitely show the relationship between CA Ⅸ expression and the migration and invasion/metastasis as well as prognosis of breast cancer, and identify CA Ⅸ as a biomarker predicting early metastasis and provide another strong evidence for CA Ⅸ target therapy.Part I CA IX Expression and its Correlation with Clinicopathological Indicators in Human Breast CancerPurpose To clarify CA IX expression and its correlation with clinicopathological indicators (including prognosis) in human breast cancer.Methods Western blot was employed to verify the expression of CA IX protein in 11 pairs of fresh breast cancer tissues and normal breast tissues. IHC was performed in 149 breast cancer samples to investigate CA IX expression and its correlation with clinicopathological indicators.Results The expression levels of CA Ⅸ in breast cancer and normal breast tissues were shown by western blot. There was almost no CA IX protein that can be detected in normal breast tissues. High expression of CA IX was observed in some breast cancer tissues. Compared to normal breast tissues, several breast cancer tissues showed significantly elevated expression levels of CA Ⅸ (p<0.001). CA Ⅸ immunoreactivity was readily detected in the cytomembrane. Only tumors showing a strong membranous staining in ≥10% or more cells were considered positive for CA IX. We did observe significant correlations of CA Ⅸ with tumor size (p= 0.019), lymph node metastasis (p= 0.006), metastatic status (p< 0.001) and clinical TNM stage (p= 0.003). Kaplan-Meier survival analysis of 149 cases revealed a correlation between higher CA Ⅸ expression levels and shorter disease-specific survival times (p < 0.001). Factors that affected survival by univariate analyses included clinical TNM stage, CA IX and distant metastasis (all p< 0.05)Conclusions Compared to normal breast tissues, several breast cancer tissues show significantly elevated expression levels of CA IX. CA IX has significant correlations with tumor size, lymph node metastasis, distant metastatic status and clinical TNM stage. CA Ⅸ is an independent biomarker in breast cancer prognosis analysis.Part Ⅱ Promotion of Migration and Invasion/Metastasis by CA Ⅸ Induced by CoCl2 in Human Breast CancerPurpose To investigate the effects of CA Ⅸ under hypoxic conditions on migration and invasion/metastasis of human breast cancer.Methods We established breast cancer cell lines hypoxic model with CoCl2. Human breast cancer cells (MDA-MB-231 and MCF7) were divided into control groups and the hypoxia groups. The hypoxia cells were exposed to CoCl2 for 24,48,72h, while control cells were cultured under normal conditions. Western blot was employed to investigate CA Ⅸ expression changes under hypoxic conditions. Migration assay was used to analyze the effects of CA Ⅸ on migration in vitro. Transwell invasion assay was utilized to evaluate the effects of CA Ⅸ on invasion.Results Western blot showed that HIF-1α protein expressions were increased in CoCl2-induced hypoxia in MDA-MB-231 and MCF7 breast cancer cell lines. Cells exposed to CoCl2-induced hypoxia after 24,48h showed no significant changes of CA Ⅸ expression levels, but the amount of CA Ⅸ protein after CoCl2 treatment of 72h was significantly increased. Transwell migration and invasion assay showed that CA Ⅸ promotes breast cancer proliferation and invasion/metastasis in vitro.Conclusions CoCl2 induced hypoxia causes an increase in expression of HIF-1α and CA Ⅸ. The elevation of CA Ⅸ tends to lag behind HIF-1α elevation, which is more obvious in earlier hypoxic period. CA Ⅸ promotes migration and invasion/metastasis in human breast cancer in vitro.