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

Kang R, Hou W, Zhang Q, Chen R, Lee YJ, Bartlett DL, Lotze MT, Tang D, Zeh HJ - Cell Death Dis (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastrointestinal Surgical Oncology, Department of Surgery, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15219, USA.

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.

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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
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fig4: 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

Mentions: Although HIF1α has a major role in the cell survival response to hypoxia,30,31 it also is associated with cell death in some instances.32 Given that the RAGE-mediated KRAS pathway is an important regulator of HIF1α signaling (Figures 2 and 3), we therefore determined whether the knock down of RAGE regulates cell survival and cell death under hypoxic conditions. Similar to our previous study,21 knockdown of RAGE and HIF1α decreased cell viability as assessed in a CCK8 assay (Figure 4a) and increased apoptosis demonstrated by enhanced caspase-3 activity (Figure 4b) following hypoxia. The RAF inhibitor (e.g., RAF265), MEK inhibitor (e.g., U0126), PI3K inhibitor (e.g., LY294002), 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) all increased hypoxia-induced cell death (Figure 4c). These findings suggest that activation of the RAGE-KRAS-HIF1α pathway under hypoxia promotes the survival of pancreatic tumor cells. Previous studies suggested that autophagy, a lysosome-mediated degradation pathway, is a major pro-survival mechanism of various tumor cells in response to hypoxia.33,34 Similarly, the knock down of key autophagy regulators (ATG5 and Beclin1) by shRNA inhibits their protein expression (data not shown) and promotes pancreatic tumor cell death under hypoxic conditions (Figure 4d), supporting a pro-survival role of autophagy in the hypoxic tumor microenvironment.35 Importantly, the knock down of RAGE and HIF1α in Panc02 cells inhibits hypoxia-induced autophagy and increases hypoxia-induced apoptosis by western blot analysis of LC3-II/LC3-I ratio (a marker of autophagy) and cleaved PARP (a marker of apoptosis) (Figure 4e). The adaptor protein p62/SQSTM1 is decreased when autophagy is induced, whereas it accumulates when autophagy is impaired.36 Consistently, the knock down of RAGE and HIF1α in Panc02 cells prevents the expected decrease in p62 levels following induction of hypoxia (Figure 4e). Moreover, MEK inhibition (e.g., U0126) and PI3K inhibition (e.g., LY294002) also inhibit autophagy (LC3-II/LC3-I ratio) and increase apoptosis (cleaved PARP) in response to hypoxia (Figure 4f). Taken together, our findings suggest that autophagic flux is enhanced under hypoxic conditions, dependent on cell survival signals generated by activation of the RAGE-KRAS-HIF1α pathway.


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

Kang R, Hou W, Zhang Q, Chen R, Lee YJ, Bartlett DL, Lotze MT, Tang D, Zeh HJ - Cell Death Dis (2014)

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
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fig4: 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
Mentions: Although HIF1α has a major role in the cell survival response to hypoxia,30,31 it also is associated with cell death in some instances.32 Given that the RAGE-mediated KRAS pathway is an important regulator of HIF1α signaling (Figures 2 and 3), we therefore determined whether the knock down of RAGE regulates cell survival and cell death under hypoxic conditions. Similar to our previous study,21 knockdown of RAGE and HIF1α decreased cell viability as assessed in a CCK8 assay (Figure 4a) and increased apoptosis demonstrated by enhanced caspase-3 activity (Figure 4b) following hypoxia. The RAF inhibitor (e.g., RAF265), MEK inhibitor (e.g., U0126), PI3K inhibitor (e.g., LY294002), 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) all increased hypoxia-induced cell death (Figure 4c). These findings suggest that activation of the RAGE-KRAS-HIF1α pathway under hypoxia promotes the survival of pancreatic tumor cells. Previous studies suggested that autophagy, a lysosome-mediated degradation pathway, is a major pro-survival mechanism of various tumor cells in response to hypoxia.33,34 Similarly, the knock down of key autophagy regulators (ATG5 and Beclin1) by shRNA inhibits their protein expression (data not shown) and promotes pancreatic tumor cell death under hypoxic conditions (Figure 4d), supporting a pro-survival role of autophagy in the hypoxic tumor microenvironment.35 Importantly, the knock down of RAGE and HIF1α in Panc02 cells inhibits hypoxia-induced autophagy and increases hypoxia-induced apoptosis by western blot analysis of LC3-II/LC3-I ratio (a marker of autophagy) and cleaved PARP (a marker of apoptosis) (Figure 4e). The adaptor protein p62/SQSTM1 is decreased when autophagy is induced, whereas it accumulates when autophagy is impaired.36 Consistently, the knock down of RAGE and HIF1α in Panc02 cells prevents the expected decrease in p62 levels following induction of hypoxia (Figure 4e). Moreover, MEK inhibition (e.g., U0126) and PI3K inhibition (e.g., LY294002) also inhibit autophagy (LC3-II/LC3-I ratio) and increase apoptosis (cleaved PARP) in response to hypoxia (Figure 4f). Taken together, our findings suggest that autophagic flux is enhanced under hypoxic conditions, dependent on cell survival signals generated by activation of the RAGE-KRAS-HIF1α pathway.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastrointestinal Surgical Oncology, Department of Surgery, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15219, USA.

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.

Show MeSH
Related in: MedlinePlus