Abstract
Cancer-specific promoter driven replication of oncolytic adenovirus (Ad) is cancer-specific, but shows low transcriptional activity. Thus, we generated several chimeric α-fetoprotein (AFP) promoter variants, containing reconstituted enhancer and silencer regions, to preferentially drive Ad replication in hepatocellular carcinoma (HCC). Modified AFP promoter, containing 2 enhancer A regions and a single enhancer B region (a2bm), showed strong and HCC-specific transcription. In AFP-positive HCCs, gene expression was 43- to 456-fold higher than those of control AFP promoter lacking enhancers. a2bm promoter was further modified by inserting multiple hypoxia-responsive elements (HRE) to generate Ha2bm promoter, which showed stronger transcriptional activity than a2bm promoter under hypoxic conditions. Ha2bm promoter-regulated oncolytic Ad (Ha2bm-d19) showed a stronger antitumor and proapoptotic effect than did a2bm promoter-regulated oncolytic Ad (a2bm-d19) in HCC xenograft tumors. Systemically administered Ha2bm-d19 caused no observable hepatotoxicity, whereas control replication-competent Ad, lacking cancer specificity (d19), induced significant hepatic damage. Ha2bm-d19 caused significantly lower expression of interleukin-6 than d19, showing that HCC-targeted delivery of Ad attenuates induction of the innate immune response against Ad. This chimeric AFP promoter enabled Ad to overcome the hypoxic tumor microenvironment and target HCC with high specificity, rendering it a promising candidate for the treatment of aggressive HCCs.
Highlights
Liver cancer is the tenth most common malignant tumor worldwide and accounts for a large fraction of overall cancer mortality[1]
All AFP promoter variants selectively induced the expression of luciferase in AFP-positive hepatocellular carcinoma (HCC) cells, whereas minimal to no luciferase activity was observed in AFP-negative cancer cells and normal cells, indicating that the newly generated AFP promoter variants expressed target genes selectively in AFP-positive HCCs
The initial screening of luciferase-expressing plasmid vectors revealed that the a2bm promoter, containing two enhancer A (EA) and one enhancer B (EB) regions upstream of the minimal AFP promoter, induced a significantly higher expression of luciferase than those expressed by other promoter variants
Summary
Liver cancer is the tenth most common malignant tumor worldwide and accounts for a large fraction of overall cancer mortality[1]. Oncolytic adenovirus (Ad) is emerging as a promising new modality for cancer treatment because it can selectively replicate and produce its progenies in cancer cells, inducing tumor cell lysis; this is a function that no anti-cancer drugs can mimic[5,6]. Several strategies, such as partial deletion of the viral genome[7,8,9] or placing the Ad E1A gene, which is essential for viral replication, under the control of tumor-specific promoters, can be used to provide Ad with tumor specificity[10,11]. The chimeric enhancer pART, which possesses four tandemly repeated T-cell factor/lymphoid enhancer factor response elements of cyclooxygenase-2 upstream of the osteocalcin nucleosome sequence signal, significantly enhances the transcriptional activity of human regenerating islet-derived 1 alpha/pancreatic stone protein/pancreatic thread protein gene[20]
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