RT Journal Article
SR Electronic
T1 Breast tumor-associated metalloproteases restrict reovirus oncolysis by cleaving the σ1 cell-attachment protein, and can be overcome by mutation of σ1
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 742478
DO 10.1101/742478
A1 Jason Fernandes
A1 Francisca Cristi
A1 Heather Eaton
A1 Patricia Chen
A1 Sarah Haeflinger
A1 Isabelle Bernard
A1 Mary Hitt
A1 Maya Shmulevitz
YR 2019
UL http://biorxiv.org/content/early/2019/08/25/742478.abstract
AB Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyoma virus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein σ1, which diminished virus binding to sialic acid-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated σ1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231 and HS578T breast cancer cell lines recapitulated σ1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers, and that breast cancer cells in by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of sialic acid (SA) levels on a panel of cancer cells showed that truncated σ1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in σ1 that prevents σ1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, equivalent toxicity as wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data shows that tumor microenvironments have the potential to reduce infectivity of reovirus.SIGNIFICANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have negative impact on proteinaceous cancer therapies such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.