The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival

Abstract

Activation of the induced receptor for advanced glycation end products (RAGE) leads to initiation of NF-kappaB and MAP kinase signaling pathways, resulting in propagation and perpetuation of inflammation. RAGE-knockout animals are less susceptible to acute inflammation and carcinogen-induced tumor development. We have reported that most forms of tumor cell death result in release of the RAGE ligand, high-mobility group protein 1 (HMGB1). We now report a novel role for RAGE in the tumor cell response to stress. Targeted knockdown of RAGE in the tumor cell, leads to increased apoptosis, diminished autophagy and decreased tumor cell survival . In contrast, overexpression of RAGE is associated with enhanced autophagy, diminished apoptosis and greater tumor cell viability. RAGE limits apoptosis through a p53-dependent mitochondrial pathway. Moreover, RAGE-sustained autophagy is associated with decreased phosphorylation of mammalian target of rapamycin (mTOR) and increased Beclin-1/VPS34 autophagosome formation. These findings show that the inflammatory receptor, RAGE, has a heretofore unrecognized role in the tumor cell response to stress. Furthermore, these studies establish a direct link between inflammatory mediators in the tumor microenvironment and resistance to programmed cell death. Our data suggest that targeted inhibition of RAGE or its ligands may serve as novel targets to enhance current cancer therapies.

Figures

Figure 1. RAGE promotes tumor cell survival following genotoxic or metabolic stress

(A) Targeted knockdown of RAGE protein following transfection with RAGE shRNA. RAGE, TLR4 and actin protein levels in Panc02 (murine) or Panc2.03 9 (human) cells transfected with nonspecific shRNA (Ctrl shRNA, “1, 3”) or RAGE shRNA (“2, 4”) at 48 h post transfection (* p 2). Cell viability was examined at 24 h (n=3, * p < 0.05 RAGE shRNA versus ctrl shRNA or pUNO versus pUNO1-RAGE). (D) Re-expression of RAGE with full length plasmid restored the chemotherapy-protective phenotype. RAGE was overexpressed in RAGE knockdown Panc02 tumor cells as indicated and then treated with chemotherapeutic agents (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml; and gemcitabine, “GM”, 100 nM). Cell viability was examined at 24 h (n=3, * p

Figure 2. Targeted knockdown of RAGE in murine and human pancreatic tumor cell lines results in increased apoptosis and decreased autophagy following treatment with chemotherapeutic agents

(A) A pan-caspase inhibitor reverses the increased caspase 3 activity and tumor cell death observed in RAGE knockdown cells. Panc02 tumor cells were pretreated with the pan-caspase inhibitor (ZVAD-FMK, 20 µM) for 1 h and then treated with chemotherapeutic agents (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml; and gemcitabine, “GM”, 100 nM). Caspase 3 activity and cell viability were examined at 24 h (n=3, * p p < 0.05 RAGE shRNA versus ctrl shRNA). (C) An autophagy inhibitor limits LC3 punctae in control and RAGE knockdown cells. Panc02 tumor cells were pretreated with the PI3K/autophagy inhibitor, wortmannin (WM) 100 nM for 1 h and then treated with chemotherapeutic agents (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml) for the indicated time. Cells were immunostained with LC3-specific antibodies. Images of 1,000 cells were analyzed to obtain the average LC3 spots by imaging cytometric [Arrayscan] analysis. Left panel is a representative picture of each condition. Inset shows a higher magnification of LC3 stain. (D) Re-expression of RAGE with full-length plasmid restores relative autophagy and apoptosis levels to baseline. RAGE was overexpressed in RAGE knockdown Panc02 tumor cells and cells were treated with chemotherapeutic agents (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml). Caspase 3 activity and LC3 spot formation were examined at 24 h (n=3, * p

Figure 3. Increased apoptosis in RAGE knockdown tumor cells is p53 dependent

(A) RAGE knockdown significantly increases phosphorylation of p53 at ser392 and decreases anti-apoptotic Bcl-2 family proteins following treatment with anti-cancer agents. Panc02 cells were treated with chemotherapeutic agents (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml) for the indicated time. Western blot analysis of protein levels is presented. Left panel is a representative picture of western blots (n=3, * P

Figure 4. Decreased autophagy in RAGE knockdown tumor cells is associated with increased levels of phosphorylated mTOR and decreased autophagasome formation

(A) RAGE knockdown results in increased phosphorylation of mTOR and decreased phosphorylation of 4E-BP1. The indicated Panc02 cells were treated with anti-cancer drugs (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml) for the given times. Western blot analysis of protein levels is shown. (B) RAGE knockdown blocks Beclin-1/VPS34 complex formation during autophagy. RAGE knockdown Panc02 cells were treated with oxaliplatin (“OX”, 160 µg/ml) for 3 h. Cell lysates were prepared for IP with anti-Beclin-1 or anti-VPS34 or IgG. The resulting immune complexes were analyzed by western blotting using antibodies to Beclin-1 or VPS34.

Figure 5. Increased sensitivity to chemotherapeutic agents, increased apoptosis and decreased autophagy in RAGE knockdown tumor cells is dependent on ATG5

(A) Western blot analysis of protein levels in Panc02 cells as indicated after knockdown RAGE, Beclin-1, and ATG5 by shRNA. (B) Depletion of ATG5, but not Beclin-1 reverses the increased chemosensitivity in RAGE knockdown cells. The indicated Panc02 cells were treated with chemotherapeutic drugs (oxaliplatin, “OX”, 160 µg/ml; melphalan, “ME”, 320 µg/ml) with or without wortmannin (100 nM) and then assayed for cell death, caspase 3 activity and LC3 punctae formation (n=3, * p

Figure 6. Expression of RAGE mediates chemoresistance in vivo and is associated with decreased apoptosis and increased autophagy

(A) RAGE knockdown tumor cells are more sensitive to oxaliplatin in vivo. C57/BLl6 mice were inoculated with 106 Panc02 tumor cells following stable transfection of control (WT) or RAGE specific shRNA (KD) and treated with oxaliplatin (“OX”, 7mg/kg) or PBS beginning at day 11 (day 11 normalized to 1). Tumors were measured twice weekly, and volumes were calculated for 38 days ( * p < 0.05 KD+OX versus WT+OX). (B) RAGE knockdown increases apoptosis following treatment with oxaliplatin (7 mg/kg) in vivo. Included here are representative images of implanted WT (control shRNA) and KD (RAGE shRNA) pancreatic tumors sections that were analyzed by TUNEL assay (green signal). Nuclear staining was done with Hoechst 33342 (blue signal). Inset shows a higher magnification of TUNEL stain. (C) RAGE knockdown decreases autophagy after treatment with oxaliplatin (7 mg/kg) in vivo. Included here are representative images of implanted WT (control shRNA) and KD (RAGE shRNA) pancreatic tumors sections that were analyzed by indirect IF staining of LC3 protein (green signal). Nuclear staining was done with Hoechst 33342 (blue signal). Inset shows a higher magnification of cells following staining with LC3.