Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival

Abstract

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumor formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signaling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signaling, and that drugs designed to target this site may be potent anti-cancer agents.

Tissue Tranglutaminase Regulates Interactions between Ovarian Cancer Stem Cells and the Tumor Niche

“…The link between TG2 and CSCs was recorded in other solid tumors. For example, TG2 upregulation was also recorded in the highly tumorigenic subpopulation of CD44 + /CD24 − breast CSCs characterized by self-renewal and mammosphere-forming capacity (13), in epidermal and glioblastoma CSCs, where it promoted spheroid proliferation and tumor formation (14,15). The connection between TG2 and chemotherapy or radiation-resistance in cancer cells (16) was attributed to activation of the NF-κB survival pathway (17,18) and of “outside-in” signaling (19), being consistent with the chemo-resistant phenotype of CSCs.…”

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“…Combination therapy induced GSCs death with subsequent significant decrease in neurosphere proliferation compared to either radiation or temozolomide treatment alone. Recent data in a different cancer model showed that the GTP-binding activity, associated with a closed conformation of TG2, but not the transamidase activity was required for epidermal CSC (ECSC) survival and spheroid formation [5]. The binding of irreversible inhibitors (NC9, VA4, and VA5) to the catalytic site of TG2 promoted a shift from its closed to an open conformation and led to a reduction in the survival and proliferation of ECSCs [5].…”

“…Recent data in a different cancer model showed that the GTP-binding activity, associated with a closed conformation of TG2, but not the transamidase activity was required for epidermal CSC (ECSC) survival and spheroid formation [5]. The binding of irreversible inhibitors (NC9, VA4, and VA5) to the catalytic site of TG2 promoted a shift from its closed to an open conformation and led to a reduction in the survival and proliferation of ECSCs [5]. The complex between TG2, fibronectin and integrins has also been proposed as a functional cancer target and inhibitors for this proteinprotein interaction are being developed [6].…”

“…Aldehyde dehydrogenases (ALDHs) comprises a family 19 evolutionary conserved NADP( + )-dependent enzymes that regulate cellular detoxification and tissue homeostasis by catalyzing the oxidation of endogenous and exogenous aldehydes into corresponding acids [5]. High ALDH activity and expression has been associated with increased cancer cell aggressiveness, metastatic potential, pluripotency and chemo-resistance in several cancer types.…”

“…TG2 expression was increased in ovarian CSCs derived from human tumors and defined by co-expression of CD44 + and CD117 + [3]. TG2 upregulation was also recorded in the highly tumorigenic subpopulation of CD44 + /CD24 -breast CSCs [4], contributing to selfrenewal, chemo-resistance and mammosphere-forming capacity and in epidermal CSCs (ECSC) where it promoted sphere formation and tumorigenicity [5].…”