A modified hTERT promoter-directed oncolytic adenovirus replication with concurrent inhibition of TGFβ signaling for breast cancer therapy

Abstract

Our laboratory is interested to develop oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we have constructed mhTERTAd.sTβRFc, a 01/07 based adenoviral vector expressing the soluble form of TGFβ receptor II fused with human Fc IgG1 (sTGFβRIIFc) gene, in which viral replication is under the control of modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTβRFc-mediated sTGFβRIIFc production would target growth factor-β (TGFβ) pathway known to contribute to the tumor progression breast cancer metastasis. We chose to use mhTERT promoter because it was found to be relatively more active (approximately 20-times) in breast cancer cells compared to normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 hrs with mhTERTAd.sTβRFc produced high levels of sTGFβRIIFc (greater than 1 μg/ml) in the medium. Breast cancer cells produced nearly 6,000-fold increase in the viral titers during 48 hrs infection period. However, mhTERTAd.sTβRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication deficient virus Ad(E1-).sTβRFc also produced high levels of sTGFβRIIFc, but under these conditions no detectable viral replication was observed. Adenoviral-mediated production of sTGFβRIIFc was shown to bind with TGFβ-1, and abolished the effects of TGFβ-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTβRFc intravenously into MDA-MB-231 human xenograft bearing mice resulted in significant inhibition of tumor growth, and production of sTGFβRIIFc in the blood. On the other hand, intravenous injection of Ad(E1-).sTβRFc did not exhibit significant inhibition of the tumor growth, but resulted in the sTGFβRIIFc in the blood, suggesting that viral replication along with sTGFβRIIFc protein production play a critical role in inducing inhibition of tumor growth. These results warrant future investigation of mhTERTAd.sTβRFc as an anti-tumor agent in vivo.

TGFβ-dependent induction of interleukin-11 and interleukin-8 involves SMAD and p38 MAPK pathways in breast tumor models with varied bone metastases potential

“…The cells were washed with serum free media before treatment with TGFβ-1 (Sigma Life Science) for one hour, and cell lysates subjected to western blot analyses using published methods. 19,22 Membranes were treated with phospho-SMAD2, phospho-SMAD3, SMAD2/3, phospho-p38 MAPK or p38 MAPK antibodies (Cell Signaling) followed by horseradish peroxidase-conjugated bovine anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). The blots were visualized by enhanced chemiluminescence substrate (Amersham Biosciences).…”

Co-expression of herpes simplex virus thymidine kinase and Escherichia coli nitroreductase by an hTERT-driven adenovirus vector in breast cancer cells results in additive anti-tumor effects

“…recently, we and other groups have shown that adenoviruses carrying the human telomerase reverse transcriptase (htert) promoter could target expression of therapeutic genes in human nasopharyngeal carcinoma cells and breast cancer cells (18,19). song et al reported that an adenovirus vector can maintain cancer cell specificity through the hTERT promoter and that the activated expression of HsV-tK can be increased by an sV40 enhancer (20).…”

Targeted Expression of BikDD Eliminates Breast Cancer with Virtually No Toxicity in Noninvasive Imaging Models

“…The human telomerase reverse transcriptase (T) is highly expressed in a majority of cancer types but not in normal cells, which allows it to be used as a tumor biomarker (15,17,18). Previously, we identified and validated the specificity of the T promoter in ovarian cancer and showed that its activity was enhanced by the VISA amplification system (15).…”

“…17,18), survivin (19,20), claudin-4 (21-23), fatty acid synthase (FASN; refs. 24,25), and b-catenin (26).…”