WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Abstract

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.

Regulation of cell signaling and apoptosis by tumor suppressor WWOX

“…WWOX triggers autophagy-inhibitory mTOR signaling and downregulates the expression of autophagy-essential proteins Atg12-Atg5, Beclin-1, and LC3-II in MTX-sensitive SCC cells (Figure 3). 42 In contrast, autophagy is highly active in MTX-resistant SCC cells (Figure 3). The failure to induce WWOX upregulation in these SCC cells has been demonstrated to be associated with chemoresistance.…”

“…Continuous intra-arterial infusion of a folate antagonist methotrexate (MTX) resulted in complete tumor regression in patients with verrucous carcinoma. 42 MTX treatment upregulates WWOX expression along with caspase activation and apoptotic cell death in both SCC tumor biopsies and cancer cell lines. 42 Upregulation of WWOX expression increases chemotherapeutic drug-induced apoptosis by dampening autophagy in MTX-sensitive SCC cancer cells.…”

“…42 MTX treatment upregulates WWOX expression along with caspase activation and apoptotic cell death in both SCC tumor biopsies and cancer cell lines. 42 Upregulation of WWOX expression increases chemotherapeutic drug-induced apoptosis by dampening autophagy in MTX-sensitive SCC cancer cells. 42 Autophagy, a conserved intracellular catabolic process, has been suggested to facilitate the survival of rapidly growing cancer cells that have outgrown their vascular supply and encounter oxygen shortage or metabolic stress.…”

“…Treatment of cells with TNF-a, UV irradiation, staurosporine, anisomycin, chemotherapeutic drugs, hypoxia induces protein phosphorylation of WWOX at Tyr33. 15,36,42 Tyr33-phosphorylated WWOX is essential for binding and stabilizing Ser46-phosphorylated p53 ( Figure 2). 15 The active WWOXp53 complexes then translocate into the nucleus where they promote apoptosis ( Figure 2).…”

“…42 Upregulation of WWOX expression increases chemotherapeutic drug-induced apoptosis by dampening autophagy in MTX-sensitive SCC cancer cells. 42 Autophagy, a conserved intracellular catabolic process, has been suggested to facilitate the survival of rapidly growing cancer cells that have outgrown their vascular supply and encounter oxygen shortage or metabolic stress. Autophagy may also help cancer cells fight off chemotherapeutic drug-induced stress responses.…”