Study On The Relationship Between Drug Resistance And Metastasis Of Breast Cancer By Tissue Array And Proteomics Methods

Objective:Drug resistance and metastasis is the major cause of treatment failure and mortality for cancer patients as an estimated 90% of all cancer patients may develop metastases or drug resistance. Previously, studies of drug resistance and metastasis generally proceeded along separate pathways of research. More recently, however, intense interest has been focused on the relationship between drug resistance and cancer metastasis. The evidence is growing rapidly that drug resistance and cancer metastasis are sometimes linked to each other: firstly, some tumor cells selected for resistance to drugs are more invasive/metastatic relative to non-resistant parental cells; secondly, in some cases, secondary (more metastatic) tumors are more resistant to chemotherapeutic drugs than their primary counterparts. All these findings are consistent with two clinical phenomena: first, metastatic tumors are more resistant to chemotherapeutic drugs than those that are nonmetastatic; second, drug resistant tumors are more invasive/metastatic relative to non-resistant parental tumors. We hypothesize that drug resistance and tumor metastasis may be inseparable events during the progression of malignant tumors, as there is a functional linkage between the two phenotypes. A better understanding of any relationship between drug resistance and cancer metastasis could lead to more effective cancer treatment.Development of drug resistance and of metastasis is complex processes because of heterogeneity of human cancers. It is unrealistic to expect any single mechanism to explain either. Traditional approaches are insufficient to describe the complex processes of drug resistance and metastasis comprehensively, so systematic approaches are needed to report global response to signal pathways in order to fully understand the mechanisms of drug resistance and cancer metastasis and to explore new targets for cancer therapy. So, tissue array and proteomics methods were used to analyze the complex processes of cancer drug resistance and metastasis in this study, our purpose is to screen some novel proteins that correlated with cancer metastasis and drug resistance and to study the relationship between multidrug resistance (MDR) and metastatic potential during the progression of malignant tumor.Methods:Part 1:1. RT-PCR and Immunohistochemical methods were used to examine the changes of expression levels of the transcription factor Twist, E-cadherin and N-cadherin in the MCF-7 breast cancer cell line and its multidrug-resistant variant, MCF-7/ADR;2. Tissue array and S-P Immunohistochemical technique methods was used to detect the expression of MDR1,MRP, LRP and GST-πcorrelated to drug resistance and the expression of MMP-2,MMP-9 and MMP-13 correlated to cancer metastasis in 263 breast cancer tissues.Part 21. Total proteins of MCF-7 and MCF-7/ADR cells were extracted and 2D PAGE based proteomics methods were used to analyze the two samples. The first dimension was carried out by IPG pH gradient isoelectric focusing system and the second dimension was separated by vertical SDS-PAGE; the gels were stained by Silver or Coommassie Brilliant Blue. The 2D PAGE maps were matched and analyzed by ImageMaster 2D platinum software to find differentially expressed proteins spots.2. Peptide Mass Fingerprint maps of differentially expressed proteins was obtained by MALDI-TOF/MS methods. 3. Bioinformatics methods were used to identify differentially expressed proteins spots and to primarily analyze the function of proteins by searching from data banks.Results:Part 11. In MCF-7 cells, the expression of E-cadherin can be detected, but there is no expression of Twist or N-cadherin. In MCF-7/ADR cells, E-cadherin expression is lost, but the expression of two other genes was significantly positive.2. The expression levels of MDR1 and LRP is correlated with lymph node status (p＜0.05); MMP-2 and MMP-9 has correlation with tumor size (p＜0.05) and MMPs all has significant correlation with clinical TNM stage and lymph node status (p＜0.01).3. The expression of MMP9 has significant correlation with MRP(p=0.004) and MDR1 (p=0.000), MDR1 expression has significant correlation with MMP13 (p=0.001), LRP expression has significant correlation with MMP2 (p=0.001).Part 21. High resolution and reproducibility of 2D PAGE technique for separation of proteins was established. The 2DE patterns were similar between the drug sensitive breast cancer cell line MCF-7 and drug resistance cell line MCF-7/ADR; The mean resolution were 868±25 spots on MCF-7 patterns and 879±19 spots on MCF-7/ADR patterns. 777 spots were matched between the two maps and the match rate was 89.6%.2. 67 proteins spots were differential expression between the two cells and 46 protein spots were up-regulation expression in MCR-7 cells and 21 protein spots were up-regulation expression in MCF-7/ADR cells; 6 proteins spots has no expression in MCF-7 cells and 2 proteins spots has no expression in MCF-7/ADR cells.3. Peptide Mass Fingerprint maps of 10 proteins spots were obtained by MALDI-TOF/MS methods successfully and 6 of the 10 proteins spots were also analyzed by MS/MS methods.4. 11 proteins were identified through the database search, 3 proteins up-regulation in MCF-7 cells were NADPH-flavin reductase, Ras-related nuclear protein and fructose-1,6-bisphosphatase; other 8 proteins up-regulation in MCF-7/ADR cells is cytidine 5'-monophosphate N-acetylneuraminic acid synthetase, Annexin A2, Ubiquitin carboxyl-terminal esterase L1, KRT1 protein, beta 2-microglobulin, kininogen 1, beta-gonadotropin and a hypothetical protein.Conclusion:1. Epithelial-Mesenchymal Transitions induced by Twist, may have a relationship with enhanced invasion and metastatic potential during the development of multidrug-resistant MCF-7/ADR breast cancer cells.2. Results of tissue array indicated that expression of MDR1 and LRP in breast cancer tissues have significant correlation with lymph node status; expression of drug resistance-related proteins has significant correlation with metastasis-related proteins. We hypothesize that drug resistance and tumor metastasis may be inseparable events during the progression of malignant tumors, as there is a functional linkage between the two phenotypes. Therefore the activity and expression of drug resistance genes in breast cancer tissues may simultaneously activate genes that lead to cancer metastasis.3. High resolution and reproducibility of 2D PAGE technique for separation of proteins and bioinformatics methods for searching of PMF from data-banks was established. 4. Primary results indicated that the differential expression proteins between MCF-7 and MCF-7/ADR cells were involved in cancer metastasis, energy metabolism and cell proliferation. The results provided the referent evidence for continue to study the relationship between drug resistance and metastasis behavior of cancer.