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

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.

The Opportunity of Precision Medicine for Breast Cancer With Context‐Sensitive Tumor Suppressor Maspin

“…It is well documented that normal breast epithelial cells MCF‐10A can form acini in three dimensional extracellular matrix (ECM) such as Matrigel. In an earlier study, we have reported the generation of MCF‐10A‐derived maspin knock‐ down stable clones [Dean et al, ]. In contrast to the acini formation by the parental MCF‐10A and scrambled siRNA‐transfected control (NC1), maspin knock‐down clones (siMas8 and siMas11) were more proliferative, formed disorganized cellular aggregates with increased infiltration into the Matrigel (Fig.…”

“…Laboratory investigations further demonstrate that the multifaceted tumor suppressive activities of maspin may derive from its multiple sub‐cellular compartmentalization. Maspin can be secreted via dual non‐classical secretory pathways as both a soluble protein and as an exosomal cargo protein [Dean et al, ]. Soluble maspin has been shown to inhibit tumor cell invasion and motility [Sheng et al, , ; Zhang et al, ], increase cell adhesion to ECM [Bernardo et al, ], block tumor‐associated ECM degradation [Cher et al, ], and retard the detachment of cancer cells from established focal adhesion sites [Yin et al, ].…”

“…Endogenously expressed maspin has been found in the nucleus and cytoplasm, and detected on the surface of cell membrane. It is also secreted free [Pemberton et al, 1997;Sternlicht et al, 1997;Bodenstine et al, 2014;Dean et al, 2017] and as an exosomal cargo protein [Dean et al, 2017]. Accumulated evidence demonstrates that maspin is a multifaceted tumor suppressor.…”

Exosomes for Diagnosis and Therapy in Gastrointestinal Cancers

“…After extraction, exosomes can be conveniently processed for physical characterization by means of Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM) [50]. In the typical experimental procedure, the obtained exosome suspension, after isolation and physical characterization, may be homogenized.…”

Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

“…We have previously reported the existence of different maspin isoforms in the cell [16,22]. Maspin is found in different cellular compartments other than nucleus, including mitochondria [23], endoplasmic reticulum-associated vesicles [2], plasma membrane [24] and exosomes [25]. Therefore, maspin isoforms may be located in different compartments, implying a diverse regulation of subcellular localization.…”