The secretion and biological function of tumor suppressor maspin as an exosome cargo protein.

Ivory Dean,Sijana H Dzinic,Alexander Kaplun,Yi Zou,M Margarida Bernardo,Shijie Sheng,James Granneman,Xiaohua Li,Guangzhao Mao,Vickie Kimler

doi:10.18632/oncotarget.13302

Abstract

Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.

Highlights

Maspin is a 42 kDa epithelial-specific tumor suppressor that predicts a better cancer prognosis, and is down-regulated in the progression of breast, prostate, lung, and esophageal squamous cancers [1,2,3,4]
To quantitatively assess the distribution of maspin in total cell lysates, vesicle depleted conditioned media (VDCM), and exosome fractions, we first performed western blotting (WB) of purified recombinant maspin produced by baculovirus-infected insect cells, rMaspin(i) [38] and constructed a working dose-response curve based on the linear detection range of 10-200 ng (R2= 0.96, Figure 1A)
To determine whether epithelial cells spontaneously secrete maspin as a soluble protein and as an exosome-associated protein, and whether the secretion of maspin is further differentially regulated in tumor cells, we prepared vesicledepleted conditioned media (VDCM) and exosome fractions from the aforementioned normal and tumor cell lines grown in serum free media

Summary

Introduction

Maspin is a 42 kDa epithelial-specific tumor suppressor that predicts a better cancer prognosis, and is down-regulated in the progression of breast, prostate, lung, and esophageal squamous cancers [1,2,3,4]. The maspin protein sequence aligns with members of the serine protease inhibitor (serpin) superfamily [12]. Maspin does not have an apparent signature sequence to direct its trafficking to specific subcellular compartments. While it is predominantly localized within the cell, in the nuclei and the cytoplasm, it can be cell-surface associated, and secreted to the medium or extracellular milieu [2, 3, 17,18,19,20]. Nuclear maspin inhibits HDAC1 and controls the expression of a small set of genes critical for epithelial differentiation [6] while extracellular maspin inhibits cell surface-associated pro-uPA [24]. Yu et al showed that upon DNA-damaging irradiation, non-small cell lung cancer H460 cells secrete maspin as an exosomal protein in a p53-dependent manner [25]

Methods

Results

Conclusion