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

C-W Tsai,T-C Huang,M-S Jan,N-S Chang,S-M Chen,P-C Hsu,Y-S Lin,M-C Sheen,F-J Lai,L-J Hsu,T-T Huang,T-H Chang,S-T Chen,H-M Sheu,W-C Chang

doi:10.1038/cddis.2013.308

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.

Highlights

The majority of anticancer drugs target rapidly proliferating cells and induce cell death by means of apoptosis
Our results showed that MTX-induced cell death in SCC-4 and SCC-15 cells in both time- and dose-dependent manners (Figures 1a and b)
Beclin-1 is a mammalian ortholog of the yeast Atg[6] that has been shown to be required for the initiation of autophagy through its interaction with class III PI3-kinase/Vps34.31 Our results showed that ectopic overexpression of WW domain-containing oxidoreductase (WWOX) suppressed protein expression of Beclin-1 in both SCC-9 and SCC-15 cells (Figure 6d)

Summary

Introduction

The majority of anticancer drugs target rapidly proliferating cells and induce cell death by means of apoptosis. MTX-induced upregulation of WWOX protein expression was accompanied by the sequential activation of caspase-9 and caspase-3 in SCC-15 cells, as evidenced by reduction of pro-caspases and increase of their cleavage forms (Figure 1h).

Results

Conclusion