Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase

Abstract

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg 340 to Ala (Mas R340A) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435-and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas R340A, enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H 2 O 2 treatment not only induced GST expression but also increased intracellular maspin/ GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H 2 O 2 -induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.Maspin, a novel serine protease inhibitor (serpin), 2 remains a promising potential therapeutic agent for a tumor suppressive effect in several cancer models (1-4). More recently, we showed that maspin expression inhibited prostate carcinoma bone tumor growth, osteolysis, and angiogenesis in a clinically relevant in vivo model (5). Consistently, differential expression of maspin has been linked to the progression of many types of human cancers. For example, in breast, colon, thyroid, lung, oral squamous, and prostate cancer, maspin expression predicts a better prognosis (6).Considering the therapeutic potential of maspin, it is critical to understand its molecular mechanisms. Three functional features of maspin are particularly noteworthy. First, an inhibitory effect of maspin on cell surface-assoc...

Tumor suppressive maspin and epithelial homeostasis

“…In search of the intracellular partners of maspin, we constructed a full-length maspin bait for yeast two-hybrid screening (using both human prostate epithelial cDNA library and a HeLa cDNA library) and identified GST (m and o isoforms), Hsp90 and HDAC1 as candidate maspin interactors [Yin et al, 2005b]. We confirmed these interactions in human prostate tissues.…”

“…The maspin/GST interaction was initially characterized [Yin et al, 2005b]. Endogenous maspin correlates with increased cellular GST activity, even though purified maspin does not affect the activity of GST in vitro.…”

“…Since extracellular maspin is efficiently internalized [Biliran and Sheng, 2001;Odero-Marah et al, 2003], it is possible that internalized maspin may initiate an insideout signaling mechanism that subsequently changes the FAC dynamics. The identification of several intracellular molecules as candidate maspin partners [Bailey et al, 2005;Yin et al, 2005b] may be of value for future research in this direction. In the meantime, it is not unreasonable to assume that the endocytosis of maspin must start with some kind of molecular interactions on the cell surface.…”

Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution

“…GSTs are also forming complexes with stress-activated protein kinases such as N-terminal c-Jun kinase and various other regulatory proteins (19,20). Additionally, GSTs inhibit oxidative stress-induced generation of reactive oxygen species and induction of vascular endothelial growth factor A through interaction with the protease inhibitor maspin (21). It is obvious that any mutations in proteins with such multifarious functions may have wide ranging consequences in cells where they are expressed.…”

Binding of Extracellular Maspin to β1 Integrins Inhibits Vascular Smooth Muscle Cell Migration

“…Nevertheless it has been shown to enter secretory vesicles (21) and is found extracellularly, in the cytoplasm and also in the nucleus (21, 22). Cytoplasmic and nuclear binding proteins for maspin have been identified (23)(24)(25), and may be responsible for its effects on proliferation and apoptosis. How secreted, extracellular maspin exerts its effects is unclear, but a function as a cell signaling ligand has been proposed (26 -28).…”