Breast Tumor-Associated Metalloproteases Restrict Reovirus Oncolysis by Cleaving the σ1 Cell Attachment Protein and Can Be Overcome by Mutation of σ1

Abstract

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 polyomavirus 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 (SA)-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 by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of 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, toxicity equivalent to that of wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data show that tumor microenvironments have the potential to reduce infectivity of reovirus. IMPORTANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have a negative impact on proteinaceous cancer therapies, such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization

“…This idea was first proposed in the setting of local viral or bacterial infections, in which draining LN innate immune responses were important for blocking systemic dissemination of pathogens ( 39 ). Extracellular LN protease activity may similarly contribute to attenuating infection, because it is known that metalloproteases can inactivate some viruses ( 40 , 41 ). However, the adaptive immune response also needs to be capable of “seeing” functional pathogen epitopes to generate useful antibody responses, motivating the necessity for a site where B cells can encounter intact antigen.…”

Heterogeneity in 2,6-Linked Sialic Acids Potentiates Invasion of Breast Cancer Epithelia

“…The bimodal expression of α2,6-Sial shown here can also be evidenced in several reports for MDA-MB-231 as well as other cell lines, but its consequence has not been investigated before. 36 − 40 The α2,6-Sial heterogeneity is counterpoised with relatively homogeneous expression in the same cells for other glycans such as α2,3-Sial, fucose, T/Tn-antigen, bisecting/biantennary complex N -glycans, and tri/tetra antennary complex N -glycans. This suggests that glycan diversity within tumor populations may be specific to the identity of monosaccharides and their linkage to preceding glycan moieties.…”

Genetic Modifications That Expand Oncolytic Virus Potency

“…Reovirus naturally infects through the enteric tract, but infections are rapidly cleared by the immune system with little-to-no symptom ( Organ and Rubin, 1998 ). In the enteric tract, reovirus exploits gut proteases to augment infection, so in Fernandes et al (2019) , we wondered what effect, if any, breast tumor proteases could have on reovirus infectivity. We discovered that breast tumor extracts decreased reovirus infectivity by 100-fold by cleaving reovirus cell attachment proteins and decreasing attachment of reovirus particles to breast tumor cells.…”