Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer

Abstract

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer

“…23, 26, 27, 28 AdEmpty does not encode a transgene. 26 AdRFP expresses the monomeric red fluorescent protein (a kind gift of Roger Tsien, University of California, San Diego 29 ) under the control of the human cytomegalovirus (CMV) enhancer/promoter and bovine growth hormone polyadenylation sequence (BGHpA).…”

“…30 AdFAST contains the p14 FAST open reading frame under regulation by the CMV enhancer/promoter and BGHpA. 23 AdFAST-HA has been previously described, 23 and is similar in structure to AdFAST, but contains an HA epitope tag on the C terminus of the FAST protein. AdFAST/RFP expresses the FAST protein and RFP under separate CMV promoters and BGHpA.…”

“…AdFAST/RFP expresses the FAST protein and RFP under separate CMV promoters and BGHpA. 23 Adβ-Gal has the E. coli β-galactosidase gene under control by the CMV enhancer/promoter. 28 Adenoviral stocks were amplified and purified as described previously.…”

“…23 We showed that expression of FAST protein from an E1-deleted Ad vector caused extensive cell fusion of A549 cells in culture, resulting in loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. Unfortunately, in the A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared with the controls.…”

“…Several studies have shown that expression of the FAST protein in cancer cells in culture can result in wide-spread syncytia formation, leading to apoptosis/cell death. 21, 22, 23 Mice injected with VSV-expressing p14 FAST protein had higher viral titers from infected tissue in vivo , suggesting a greater ability to spread and replicate compared with the control virus. 24 Co-administration of an oncolytic VSV armed with the p14 FAST protein increased oncolytic vaccinia virus titers and synergistically promoted cancer cell death in vitro.…”