623. Transcriptional Targeting of Breast Cancer Employing the Heparanase Promoter

Abstract

Breast cancer is a leading cause of death in the United States. Despite some advances in treatment of primary breast cancer, patients with metastatic disease still have no chance of cure and conventional treatment modalities remain palliative. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced or metastatic breast cancer. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. A critical factor determining the utility of Ad vectors for cancer gene therapy is the selectivity of their transgene expression in cancer cells. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells, especially the liver. However, currently available TSPs used in the context of Ad-based breast cancer targeting, are generally not tumor-specific but tissue specific. In this context, Heparanase (HPR), a heparan sulfate-specific endo-β-D-glucuronidase, is highly overexpressed in human cancers including breast cancer and has been shown to play an important role in tumor metastasis. In contrast, HPR expression is low or undetectable in differentiated, healthy tissue. To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancer, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control. Biodistribution and liver tropism was evaluated after i.v. virus injection into a mouse model. Quantitative RT-PCR reveals a 4.5 – 44.6 fold, (p<0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. In primary breast cancer patient samples the average HPR mRNA copy number was significantly increased (90 fold, p<0.05). When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5% – 12.7% compared to CMV) and primary breast cancer patient samples (8.8%– 14.4%), whereas activity in normal breast cells (1% compared to CMV) was low. The mouse liver toxicity and biodistribution profile of AdHPR.luc is significantly (2.3 fold, p<0.05) repressed compared to AdCMV.luc. In conclusion, we have identified the HPR promoter as a candidate for transcriptional targeting of breast cancer. In an Ad backbone, this promoter appears to be highly active in human breast cancer cells. These findings place the HPR pathway as a target for the development of breast cancer directed gene therapy strategies.