Enhanced Gene Expression in Breast Cancer Cells in Vitro and Tumors in Vivo

Abstract

Gene therapy clinical trials for cancer frequently produce inconsistent results. Some of this variability could result from differences in transcriptional regulation that limit expression of therapeutic genes in specific cancers. Systemic liposomal delivery of a nonviral plasmid DNA showed efficacy in animal models for several cancers. However, we observed large differences in the levels of gene expression from a CMV promoter-enhancer between lung and breast cancers. To optimize gene expression in breast cancer cells in vitro and in vivo, we created a new promoter-enhancer chimera to regulate gene expression. Serial analyses of gene expression data from a panel of breast carcinomas and normal breast cells predicted that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter is highly active in breast cancers. Furthermore, GAPDH is up-regulated by hypoxia, which is common in tumors. We added the GAPDH promoter, including the hypoxia enhancer sequences, to our in vivo gene expression plasmid. The novel CMV-GAPDH promoter-enhancer showed up to 70-fold increased gene expression in breast tumors compared to the optimized CMV promoter-enhancer alone. No significant increase in gene expression was observed in other tissues. These data demonstrate tissue-specific effects on gene expression after nonviral delivery and suggest that gene delivery systems may require plasmid modifications for the treatment of different tumor types. Furthermore, expression profiling can facilitate the design of optimal expression plasmids for use in specific cancers.

Mutant Bik expression mediated by the enhanced minimal topoisomerase IIα promoter selectively suppressed breast tumors in an animal model

“…36 We selected the lung fibroblast cell line as a representative normal cell line for comparison of promoter activity in this quick screening because lung is the major nonspecific targeted organ when a gene is delivered intravenously by cationic liposome. [14][15][16][17] On average, the topoIIa promoter possessed the highest level of activity in breast cancer cells and the lowest level of activity in normal or nonmalignant cell lines, suggesting that it has the highest degree of breast cancer specificity. The other promoters had either a lower level of activity in breast cancer cells (ceruloplasmin and transferrin receptor) or a higher level of activity in normal cells (B-myb).…”

“…50 When the luciferase gene is delivered by liposome through tail vein injection in a mouse model, however, the principal sites of luciferase activity are the lung, heart and liver, and the luciferase activity in lung per microgram of tissue protein is one to two order higher than that in any other organs. [14][15][16][17] This fact could reflect that gene delivery efficiency depends not only on the interstitial retention, but also on the uptake of tissues, nuclear transport of DNA and tissuespecific promoter activity. 51 In this study, a similar biodistribution pattern was observed when the DOTAP: Chol-CMV-luc complex was injected through the tail vein of tumor-bearing mice (Figure 4d, upper panels).…”

“…When cationic liposome is used to deliver CMV promoter-driven reporter gene systemically, significant uptake of the DNA construct occurs in lung and heart, resulting in a high level of reporter activity in these organs. [14][15][16][17] Undesired gene expression in normal organs may cause side effects in cancer gene therapy. 10,11 Strategies to reduce such risk for liposomal gene therapy include the adoption of a promoter that induces a higher level of gene expression in tumor cells and a lower level in normal cells, thus decreasing undesired gene expression secondary to systemic treatment.…”

“…To accelerate the clinical use of this system, we adopted materials that have already been tested in clinical trials, including backbone DNA vector pUK21 5,22 and extruded DOTAP:Chol liposome (Human Gene Transfer Protocol 0201-513, NIH Genetic Modification Clinical Research Information System). 14,15 As a first step, we determined that the topoisomerase IIa (topoIIa) promoter is specifically activated in breast cancer cells, and that its cis-element, the inverted CCAAT box (ICB), [23][24][25] mediates this specific activity. To enhance its basal activity level, we linked the minimal topoIIa promoter harboring ICB1 with the enhancer sequence from the CMV promoter.…”

Therapeutic effect of intravenous delivery of lipoplexes containing the interferon-β gene and poly I: poly C in a murine lung metastasis model

“…Recently, various types of promoters have been constructed, which shows higher levels of gene expression than CMV promoter. 52,53 In addition to the promoter, other transcriptional regulatory elements, such as intron and polyadenylation signal sequence, are also important. 54 The use of optimized transgene expression cassette would make lipoplexmediated gene therapy more efficient.…”