Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth

Abstract

We have previously reported that infection with the non-pathogenic, tumor suppressive, wild-type adeno-associated virus type 2 (AAV2) inhibited proliferation of breast cancer-derived lines representing both weakly invasive (MCF-7 and MDA-MB-468), as well as aggressive (MDA-MB-231) cancer types. AAV2-induced death occurred via targeting pathways of apoptosis and necrosis. In contrast, normal human mammary epithelial cells were unaffected upon AAV2 infection. The current study characterizes AAV2 infection and subsequent death of the highly aggressive, triple-negative (ER⁻/PR⁻/HER2⁻) MDA-MB-435 cell line derived from metastatic human breast carcinoma. Monolayer MDA-MB-435 cultures infected with AAV2 underwent complete apoptotic cell death characterized by activation of caspases -7, -8, and -9 and PARP cleavage. Death was further correlated with active AAV2 genome replication and differential expression of viral non-structural proteins Rep78 and Rep52. Cell death coincided with increased entry into S and G₂ phases, upregulated expression of the proliferation markers Ki-67 and the monomeric form of c-Myc. Expression of the p16^INK4, p27^KIP1, p21^WAF1, and p53 tumor suppressors was downregulated, indicating marked S phase progression, but sharply contrasted with hypo-phosphorylated pRb. In parallel, MDA-MB-435 breast tumor xenografts which received intratumoral injections of AAV2 were growth retarded, displayed extensive areas of necrosis, and stained positively for c-Myc as well as cleaved caspase-8. Therefore, AAV2 induced death of MDA-MB-435 xenografts was modulated through activation of caspase-regulated death pathways in relation to signals for cell cycle controls. Our findings provide foundational studies for development of novel AAV2 based therapeutics for treating aggressive, triple-negative breast cancer types.

Keywords: :

• adeno-associated virus type 2
• AAV2
• triple-negative breast cancer
• MDA-MB-435 cells
• apoptosis
• cell cycle
• Rep proteins
• c-Myc
• breast tumor xenografts
• nude mice

10.4161/cbt.29172

Disclosure of Potential Conflicts of Interest

The work described in this manuscript is part of US Patent Application No. 8 080 240 applied for by The Pennsylvania State University Intellectual Property Office.

Authors’ Contributions

S.A. and C.M. conceptualized the experimental design and drafted the manuscript. S.A. performed all experiments. B.S.B. performed the western blots for cell cycle analysis. M.I. performed the immuno-histochemical staining of the tumor tissues. M.J.C. performed statistical analysis of the tumor growth curves.

Acknowledgments

We thank D. Welch and A. Mastro for kindly supplying the MDA-MB-435 cell line. We thank Lynn Budgeon for excellent technical assistance with studies of tumor histology. This work was supported by a grant from the Pennsylvania Breast Cancer Coalition to C.M.