Abstract 862: Purified Recombinant Maspin is Processed Through the Endosomal Pathway in Breast Cancer Cells.

Abstract Maspin (Mammary Serine Protease Inhibitor), a gene involved in mammary gland development and morphogenesis, has been shown to act as a tumor suppressor in multiple cancers by inhibiting the aggressive cancer cell phenotype. Recombinant Maspin (rMas) added to cell culture media is internalized by cancer cells and capable of recapitulating many of the effects of endogenously re-expressed Maspin in Maspin-null cancer cells, including the inhibition of cell invasion, migration and induction of angiogenesis. Importantly, these effects are mediated by purified rMas without the need for post-translational modifications prior to application, increasing the future therapeutic potential for utilizing rMas as an adjuvant therapy. However, the therapeutic use of rMas is limited by its rapid clearance from cancer cells, and many of the molecular mechanisms regarding rMas internalization and processing remain to be determined. Building on preliminary data, we hypothesized that rMas is processed by the endosomal pathway. We demonstrate localization of rMas in Rab9 late-endosomes and Lamp-1 positive lysosomes following treatment of 10 μg/ml rMas in BT549, Hs578T and MDA-MB-231 human breast cancer cell lines. rMas internalization was reduced when incubated at 4oC, and increased at temperatures of 20oC and 37oC, with the highest rates of internalization at 37oC, indicative of endocytosis. Furthermore, depletion of K+ decreased rMas internalization in Hs578T and MDA-MB-231, suggestive of clarthrin-mediated endocytosis. Our data show that rMas is capable of acting at the membrane to mediate effects on invasion and migration, but may not be functional once internalized due to rapid degradation by the lysosome. Interestingly, this may provide a plausible explanation for previously reported data in which rMas was not able to reproduce the effects of endogenous Maspin re-expression on cancer cell apoptosis sensitivity, which require cytosolic and mitochondrial mechanisms. Our data provide evidence that strategies which induce endosomal escape of rMas will intensify its anti-tumor effects on cancer cells, and further enhance its potential therapeutic applications. Citation Format: Thomas M. Bodenstine, Richard E. B. Seftor, Elisabeth A. Seftor, Zhila Khalkhali-Ellis, Nicole A. Samii, J. Cesar Monarrez, Philip A. Pemberton, Mary J. C. Hendrix. Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2013-862