| Breast cancer is the most commonly diagnosed cancer in women worldwide. Theincidence of breast cancer continues to rise in recent 20 years, which is inferior touterine cervix cancer in China. There are four major modalities of treatment ofdisease in breast cancer: (1) surgery, (2) chemotherapy, (3) immunotherapy, and (4)radiation. Although the overall prognosis is excellent for primary breast cancerdiagnosed early and treated by adequate therapies, those with advanced, recurrent ormetastatic disease still have a poor chance of cure. Clearly, novel therapies for breastcancer are required. Gene therapy might be novel therapies for the advanced,recurrent or metastatic breast cancer, which transfers a therapeutic gene for mutationcompensation, immunopotentiation, or prodrug activation. To date, gene therapyapproaches have not realized a significant therapeutic end point in human clinicaltrails. Poor tumor transduction efficiency and nonspecific transcription are key factorslimiting realization of the potential of cancer gene therapy. To some extent, vectorsare more important to realize the goal.Adenovirus is widely used in the therapy of breast cancer. Various approacheshave been developed to enhance transductional and transcriptional targeting strategies.To this end, transductional targeting strategies have attempted to reengineer viraltropism such that target cell binding predicates specificity. In parallel, strategies havebeen developed to enhance the transcription selectivity of adenovirus for tumor cellsby limiting ectopic expression in non-tumor cells, thus limiting treatment-associatedtoxicities. Transcriptional targeting strategies employ the use of a tissue specificpromoter (TSP) to restrict transgene expression or viral replication to tumor cells. Theideal TSP for breast cancer would exhibit the widest differential between 'tumoron/liver off expression profiles, which is key to ablation of liver toxicity fromectopically localized adenovirus (Ad). Numerous tissue- or tumor-specific promoters(TSP) have been explored in adenovirus. Thus, to achieve the specificity of transgene expression required in the context of breast cancer gene therapy, it is necessary toevaluate promoters.CXCR4 is a G-protein coupled receptor, which encodes 352 amino acids. Mulleret al have demonstrated that CXCR4 expression is markedly upregulated in breastcancer, furthermore, SDF-1a-CXCR4 axis playing a key role in progression tometastasis. Survivin is a novel member of the inhibitor of apoptosis (IAP) proteinfamily, which plays an important role in the survival of cancer cells and theprogression of malignancies. Survivin expression is found during embryonic and fetaldevelopment and in different tumors, but is undetectable in terminally differentiatedadult tissue. Thus, the overexpression of CXCR4 and Survivin gene indicate that theymight be used as TSPs in breast cancer. CXCR4 promoter has been identified in renalcancer and melanoma, while Survivin has been used a TSP in glioma, liver cancer andgastric cancer. However, the same promoter exhibits different activity in differentcancer cells, it is necessary to evaluate them prudently. The aim of our research workis to evaluate and compare the usefulness of the two promoters for use in Ad-basedgene therapy applications for breast cancer.Quantitative RT-PCR was used to examine gene expression profiles of the twopromising target genes CXCR4 and Survivin in breast cancer cell lines which havedifferent biological properties. The levels of CXCR4 and Survivin mRNA in breastcancer cells are higher compared with HBL 100. The expression of CXCR4 mRNA is:MDA-MB-231＞MCF7＞T-47D＞MDA-MB-435＞HBL100. In MDA-MB-231,which is a high metastatic cell line, mRNA of CXCR4 increased more sharply thanMCF7 and T-47D which are low metastic cell lines. The expression of SurvivinmRNA is: T-47D＞MDA-MB-435＞MCF7＞MDA-MB-231＞HBL100, in whichMCF7 and MDA-MB-435 show the comparative expression. However, there isnotable difference between MCF7 and MDA-MB-231 which both areadenocarcinoma (P＜0.05), so do T-47D and MDA-MB-435 which are both ductalcarcinoma (P＜0.05). Overall, CXCR4 is related to metastasis of breast cancer, whileSurvivin is a not associated with the pathologic type. Taken together, CXCR4 and Survivin are transcriptionally upregulated in breast cancer cells, which indicate thepotential utility for transcriptional targeting breast cancer.Furthermore, 279bp of CXCR4 and 262bp of Survivin minimal promoters werePCR amplified and cloned into the reporter plasmid pEGFP-1 and pGL3-Basic. Theconstructs pEGFP/CXCR4, pEGFP/Survivn, pGL3B/CXCR4 and pGL3B/Survivinwere transfected into breast cancer cell and HBL 100 respectively. The expression ofEGFP and luciferase were investigated respectively. CXCR4 and Survivin genepromoter show the high transcriptional activity in breast cancer cells compared withHBL 100. CXCR4 promoter exhibits highest activity inMDA-MB-231(RLU=35.21%), while Survivin promoter is the most active inMCF7(RLU=32.72%). In the same breast cancer cell, CXCR4 promoter showstronger activity than Survivin promoter in MDA-MB-231, while weaker thanSurvivin promoter in MCF7 and T-47D. The high transcriptional activity of CXCR4and Survivin gene promoter in breast cancer cells indicates their potential utility as anovel candidate for transcriptional targeting of breast cancer.The utility of the CXCR4 and Survivin promoter in recombinant adenovirus wasevaluated in breast cancer cells. First, recombinant adenoviral plasmids (reAds)which are pAdGL3BCXCR4, pAdGL3BSurvivin and pAdGL3BCMV, wereconstructed with improved two-step homologous recombination protocol, wherebythe human CXCR4 promoter (or Survivin promoter, cytomegalovirus promoter)drives the expression of a reporter luciferase gene. Then recombinant adenovirusreAdGL3BCXCR4, reAdGL3BSurvivin and reAdGL3BCMV were packaged inAD293 cells. Next, luciferase activites were measured in multiple breast cancer celllines after three different titers of TCID50 infection with reAdGL3BCXCR4,reAdGL3BSurvivin and reAdGL3BCMV respectively. The CXCR4 and survivinpromoter were highly active in breast cancer cells compared with normal breast cellswith a dose-dependent manner. CXCR4 promoter exhibited higher activities thanSurvivin promoter in MDA-MB-231, while lower in MCF7 and T-47D. Furthermore,CXCR4 promoter had huge variation of luciferase activities in MCF7, T-47D and MDA-MB-231 cell lines. Finally, biodistribution studies in mice demonstrated moredramatic repression of the Survivin promoter in the liver, which was 1/87.46 ofCXCR4 promoters.Taken together, the survivin promoter appears to be a superior TSP for breastcancer targeting among them because it had high activity in established cell lines withlower variation and lower activity in liver. |
