CD44/Prion Protein Interact And Influence The Invasion/Metastasis, Proliferation And Drug Resistance Of Human Breast Cancer

IntroductionThe incidence of breast carcinoma is accounting for7%-8%of various malignant tumors, which is one of the major causes of death in women. In recent years, there has been great development in the treatment of breast cancer, which includes the chemotherapy after radical mastectomy and neoadjuvant chemotherapy (NAC). But during the chemotherapy, patients become resistant to some chemotherapeutic drugs. The multidrug resistance (MDR) is one of the problems in tumor therapy, and the invasive/metastatic ability of MDR cells is strengthened compared with their parental cells. It is of great significance in the investigation of the molecular mechanism related to MDR and invasion/metastasis in tumor, which can find target proteins to guide the clinical chemotherapy.The purpose of our study is to investigate the molecular mechanism of MDR and invasion/metastasis in MDR cells and sensitive cells, and find target proteins to guide the clinical chemotherapy. Therefore, this ivestigation is important to know the biological properties of tumor and has great clinical value and social benefits.Part I CD44/PrPc interact and regulate the migration, invasion/metastasis and proliferation in multidrug resistant breast cancer cellsPurpose To investigate CD44/PrPc interaction and their regulation of migration, invasion/metastasis and proliferation in MDR breast cancer cellsMethods We first analyzed the expression of CD44and PrPc using quantitative real-time PCR and immunoblotting in the MDR cell lines (MCF7/Adr and H69AR) and their parental sensitive cell line (MCF7and H69). Then we analyzed the cellular localization of PrPc and CD44in MCF7/Adr cells by confocal microscopy. We detected the expression levels of CD44, PrPc, EGFR, CD147and MMPs using immunoblotting in MCF7/Adr cells transfected with CD44siRNA or PrPc siRNA. The migration and invasion of MCF7/Adr cells transfected with CD44siRNA or PrPc siRNA was detected by a transwell assay with or without matrigel. We assessed the growth capacity of the CD44-silenced or PrPc-silenced MCF7/Adr cells using a CCK8assay and the expression level of the PI3K/Akt signaling pathway and cell-cycle-related proteins by immunoblotting.Results The mRNA and protein expression of CD44and PrPc was higher in MDR cell lines (MCF7/Adr and H69AR) than in sensitive cell lines (MCF7and H69). In MCF7/Adr cells, both CD44and PrPc located in the cytomembrane and interacted with each other. After transfected with CD44siRNA or PrPc siRNA in MCF7/Adr cells, the ability of migration, invasion and prolifertation was down-regulated, accompanied with the change of expressions of EGFR, CD147, MMPs and cell-cycle-related proteins.Conclusions In MDR breast cancer cells MCF7/Adr, there exists the interaction between CD44and PrPc. After transfected with CD44siRNA or PrPc siRNA in MCF7/Adr cells, the ability of migration, invasion and prolifertation was down-regulatedPart Ⅱ CD44/PrPc involvement in drug resistance in multidrug resistant breast cancer cellsPurpose To investigate the effect of CD44/PrPc on drug resistance in MDR breast cancer cells and its mechanismMethods We detected the expression level of CD44and PrPc in MCF7/Adr cells incubated with P-gp sustrates or non P-gp substrates using immunoblotting. The invasion ability was assessed in CD44/PrPc-silenced MCF7/Adr cells incubated with taxol or bleomycin by transwell chamber assay. Then the expression of CD44, PrPc, EGFR and CD147was analyzed by immunoblotting. We also detected the expression of P-gp, MRP1and ABCG2in CD44/PrPc-silenced MCF7/Adr cells.Results When the MCF7/Adr cells were cultured in different concentrations of P-gp substrates, the expression levels of CD44and PrPc were increased obviously and this increase was not dose-dependent. In the MCF7cells, we didn’t observe the similar phenomenon. When the CD44-silenced MCF7/Adr cells were cultured in the P-gp substrate taxol, their invasive ability was restored with increasing concentrations of this drug. Consistent with the observations above, the expression levels of CD44, PrPc, EGFR and CD147again increased. However, when the cells were cultured in bleomycin (non-P-gp substrate) as a control, their invasion remained inhibited. In CD44-silenced or PrPc-silenced MCF7/Adr cells, P-gp was decreased, whereas MRP1and ABCG2were not significantly changedConclusions In MCF7/Adr cells, P-gp substrates can up-regulate the expression of CD44and PrPc. The knock down of CD44cannot completely inhibit the invasion of MCF7/Adr cells.Part III The expression of CD44and PrPc in breast cancer tissue samples and the relationship between CD44/PrPc and neoadjuvant chemotherapyPurpose To investigate whether the expression of CD44and PrPc affects the outcomes of clinical chemotherapiesMethods IHC was performed in98breast carcinoma samples (including49pre-NAC samples and49post-NAC samples) to investigate CD44and PrPc expression and their correlation with clinical chemotherapy.Results The CD44immunoreactivity was detected mainly in the cytomembrane, and PrPc was observed predominantly in the nucleus. We observed a significant correlation between the CD44expression and the PrPc expression (r=0.411, p=0.003) in the post-NAC cases; however, CD44did not significantly correlate with PrPc in the pre-NAC cases (r=0.095, p=0.519). Moreover, we found that the CD44expression was remarkably increased in the post-NAC samples of Group2(which exhibited no response to NAC treatment) compared with the pre-NAC samples of Group2using the paired-samples t-test (p=0.040). PrPc yielded consistent results (p=0.007). In the post-NAC samples of Group1(which exhibited a partial response to treatment), the CD44expression was not obviously changed (p=0.669); by contrast, the PrPc expression was dramatically reduced (p=0.014).Conclusions The up-regulation of the CD44and PrPc expression is linked to treatment failures of clinical neoadjuvant chemotherapy.Part Ⅳ A preliminary study of miR-130a in multidrug resistant breast cancer cellsPurpose To clarify miR-130a expression in MDR cancer cells and sensitive cancer cells, and predict the target gene of miR-130aMethods We detected miR-130a expression in three kinds of MDR cancer cells and their sensitive cancer cells using realtime-PCR. The target gene of miR-130a was predicted by searching on website. Then we assessed the target gene expression in the cancer cells using realtime-PCR. Lentivirus infected MCF7/Adr cells.Results The expression of miR-130a was higher in MDR cancer cells MCF7/Adr and H69AR than in sensitive cancer cells MCF7and H69. On the other hand, the predicted target gene PTEN was decreased in MCF7/Adr and H69AR cells, compared with their sensitive cancer cells. After72h of infected with Lentivirus in MCF7/Adr cells, green fluorescence was observed and miR-130a was down-regulated.Conclusions MiR-130a was overexpressed in MDR cancer cells, while its predicted target gene PTEN was lowly expressed. Lentivirus infects with MCF7/Adr cells and down-regulates the expression of miR-130a.