RAGE is essential for oncogenic KRAS-mediated hypoxic signaling in pancreatic cancer

Abstract

A hypoxic tumor microenvironment is characteristic of many cancer types, including one of the most lethal, pancreatic cancer. We recently demonstrated that the receptor for advanced glycation end products (RAGE) has an important role in promoting the development of pancreatic cancer and attenuating the response to chemotherapy. We now demonstrate that binding of RAGE to oncogenic KRAS facilitates hypoxia-inducible factor 1 (HIF1)α activation and promotes pancreatic tumor growth under hypoxic conditions. Hypoxia induces NF-κB-dependent and HIF1α-independent RAGE expression in pancreatic tumor cells. Moreover, the interaction between RAGE and mutant KRAS increases under hypoxia, which in turn sustains KRAS signaling pathways (RAF-MEK-ERK and PI3K-AKT), facilitating stabilization and transcriptional activity of HIF1α. Knock down of RAGE in vitro inhibits KRAS signaling, promotes HIF1α degradation, and increases hypoxia-induced pancreatic tumor cell death. RAGE-deficient mice have impaired oncogenic KRAS-driven pancreatic tumor growth with significant downregulation of the HIF1α signaling pathway. Our results provide a novel mechanistic link between NF-κB, KRAS, and HIF1α, three potent molecular pathways in the cellular response to hypoxia during pancreatic tumor development and suggest alternatives for preventive and therapeutic strategies.

Figure 1

Hypoxia increases RAGE expression in an NF-κB-dependent and HIF1α-independent manner in pancreatic tumor cells. (a and b) Indicated pancreatic tumor cell lines were treated with CoCl₂ (a) or 1% O₂ (b) for 24 h. Western blot analyzed expression of RAGE, HIF1α, and other indicated proteins. (c and d) Panc02 cells were transfected with control shRNA (c) p65 shRNA (c) control siRNA (d) and HIF1α siRNA (d) for 48 h, and then treated with 1% O₂ for 24 h. The indicated protein levels were analyzed by western blot. (e and f) In parallel, the transcriptional activity of NF-κB and HIF1α (e) and RAGE mRNA level (f) were assayed (n=3, *P<0.05). (g) Panc02 cells were treated with 1% O₂ with or without NF-κB inhibitor (ammonium pyrrolidinedithiocarbamate (AP, 100 μM)) and Bay 11–7082 (Bay, 10 μM) and HIF1α inhibitor (methyl 3-((2-(4-(2-adamantyl)phenoxy)acetyl)amino)-4-hydroxybenzoate, 10 μM) for 24 h. The protein level of RAGE was analyzed by western blot. AU=arbitrary unit

Figure 2

NF-κB-mediated RAGE expression positively regulates HIF1α signaling under hypoxia in pancreatic tumor cells. (a) Indicated pancreatic tumor cell lines were transfected with control shRNA and RAGE shRNA for 48 h and then treated with 1% O₂ for 24 h. Western blot was used to analyze the expression of RAGE, HIF1α, and HIF1α-target proteins. (b–d) In parallel, nuclear HIF1α level (b and c) and the transcriptional activity of HIF1α (d) were assayed (n=3, *P<0.05). Bar=10 μm. (e and f) Panc02 cells were transfected with indicated shRNA and cDNA for 48 h and then treated with 1% O₂ for 24 h. The indicated protein levels were analyzed by western blot (e). In parallel, the transcriptional activity of HIF1α (f) was analyzed (n=3, *P<0.05). AU=arbitrary unit

Figure 3

RAGE-mediated KRAS pathway activation promotes hypoxia-induced HIF1α expression and activity in pancreatic tumor cells. (a and b) Indicated pancreatic cancer cells were treated with 1% O₂ for 24 h. The interaction between RAGE and KRAS were assayed by immunoprecipitation (a) and image analysis (b). Bar=10 μm. (c) Panc02 and PANC-1 cells were transfected with control shRNA and RAGE shRNA for 48 h, and then treated with 1% O₂ for 24 h. Western blot was used to analyze the expression of p-AKT, AKT, p-ERK1/2, and ERK1/2. Relative p-AKT and p-ERK1/2 levels were quantified (n=3, *P<0.05). (d and e) Panc02 cells were treated with 1% O₂ for 24 h with or without potential RAF inhibitor (e.g., RAF265, 1 μM), MEK inhibitor (e.g., U0126, 10 μM), PI3K inhibitor (e.g., LY294002, 10 μM), and AKT inhibitor (e.g., 1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate, 25 μM). The protein expression (d) and transcriptional activity (e) of HIF1α were assayed (n=3, *P<0.05 versus hypoxia group). (f and g) Normal or RAGE-overexpressed Panc02 cells were treated with 1% O₂ for 24 h with or without MEK inhibitor (e.g., U0126, 10 μM) and AKT inhibitor (e.g., 1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate, 25 μM). The indicated protein expression (d) and transcriptional activity of HIF1α (e) were assayed (n=3, *P<0.05). AU=arbitrary unit

Figure 4

Hypoxia-induced autophagy via the RAGE-KRAS-HIF1α pathway is a survival mechanism in pancreatic tumor cells. (a and b) Indicated Panc02 cells were treated with 1% O₂ for 24 h and cell viability (a) and caspase-3 activity (b) were then assayed (n=3, *P<0.05). (c) Panc02 cells were treated with 1% O₂ for 24 h with or without potential RAF inhibitor (e.g., RAF265, 1 μM), MEK inhibitor (e.g., U0126, 10 μM), PI3K inhibitor (e.g., LY294002, 10 μM), and AKT inhibitor (e.g., 1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate, 25 μM). Cell viability was assayed (n=3, *P<0.05). (d) Panc02 cells were transfected with control shRNA, ATG5 shRNA, and Beclin1 shRNA for 48 h, and then treated with 1% O₂ for 24 h. Cell viability was assayed (n=3, *P<0.05). (e and f) Indicated Panc02 cells were treated with 1% O₂ for 24 h with or without MEK inhibitor (e.g., U0126, 10 μM) and PI3K inhibitor (e.g., LY294002, 10 μM). Protein levels were assayed by western blot

Figure 5

Depletion of RAGE in mice impairs KRAS-mediated HIF1α signaling in vivo. (a) Western blot and (b) immunohistochemistry analysis of indicated protein expressions from pancreatic specimens at 18 weeks of age (quantitative analysis drawn from five separate high power fields, *P<0.05). Bar=100 μm. Representative positive cells were marked by red arrows

Figure 6

Hypoxia promotes pancreatic cancer initiation, progression, and metastasis through the activation of NF-κB-RAGE-KRAS-HIF1α pathway