Limits...
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.

Show MeSH

Related in: MedlinePlus

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4237264&req=5

fig1: 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

Mentions: To evaluate whether RAGE is induced by hypoxia, we first treated two individual pancreatic tumor cell lines (mouse Panc02 and human Panc2.03) with cobalt chloride 2 (CoCl2), a widely used chemical inducer of hypoxia.26 As expected,27 CoCl2 prevented HIF1α degradation and increased HIF1α expression. Similarly, the expression of the HIF1a target genes BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and BNIP3L/NIX were significantly increased following CoCl2 treatment in pancreatic tumor cells (Figure 1a).26 In contrast, non-target genes (e.g., high mobility group box 1 (HMGB1) and cyclinD1) did not change (Figure 1a). Interestingly, the expression of RAGE was increased following CoCl2 (Figure 1a) or hypoxic (1% O2) treatment (Figure 1b) in multiple pancreatic tumor cell lines, suggesting a potential role of RAGE in the response to hypoxia. Several transcription factors, including NF-κB and HIF1α, are required for upregulated RAGE expression in response to stress in cancer and non-cancer cells.20,28,29 To explore whether NF-κB and HIF1α are involved in hypoxia-induced RAGE expression in pancreatic tumor cells, we knocked down NF-κB p65 and HIF1α in Panc02 cells by specific shRNA (Figure 1c) and siRNA (Figure 1d), respectively. Suppression of NF-κB p65 and HIF1α expression significantly limited hypoxia-induced transcriptional activity of both genes (Figure 1e). As expected, suppression of HIF1α inhibited hypoxia-induced BNIP3 expression (Figure 1d). However, suppression of NF-κB p65, but not HIF1α, inhibited hypoxia-induced expression of RAGE protein (Figures 1c and d) and mRNA expression (Figure 1f). Moreover, the NF-κB inhibitor (e.g., ammonium pyrrolidinedithiocarbamate (AP) and Bay 11-7082 (Bay)), but not the HIF1α inhibitor (e.g., methyl 3-((2-(4-(2-adamantyl)phenoxy)acetyl)amino)-4-hydroxybenzoate), inhibited hypoxia-induced RAGE protein (Figure 1g) and mRNA (data not shown) expression. These findings suggest that hypoxia-induced RAGE expression is NF-κB-dependent and HIF1α-independent.


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 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
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237264&req=5

fig1: 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
Mentions: To evaluate whether RAGE is induced by hypoxia, we first treated two individual pancreatic tumor cell lines (mouse Panc02 and human Panc2.03) with cobalt chloride 2 (CoCl2), a widely used chemical inducer of hypoxia.26 As expected,27 CoCl2 prevented HIF1α degradation and increased HIF1α expression. Similarly, the expression of the HIF1a target genes BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and BNIP3L/NIX were significantly increased following CoCl2 treatment in pancreatic tumor cells (Figure 1a).26 In contrast, non-target genes (e.g., high mobility group box 1 (HMGB1) and cyclinD1) did not change (Figure 1a). Interestingly, the expression of RAGE was increased following CoCl2 (Figure 1a) or hypoxic (1% O2) treatment (Figure 1b) in multiple pancreatic tumor cell lines, suggesting a potential role of RAGE in the response to hypoxia. Several transcription factors, including NF-κB and HIF1α, are required for upregulated RAGE expression in response to stress in cancer and non-cancer cells.20,28,29 To explore whether NF-κB and HIF1α are involved in hypoxia-induced RAGE expression in pancreatic tumor cells, we knocked down NF-κB p65 and HIF1α in Panc02 cells by specific shRNA (Figure 1c) and siRNA (Figure 1d), respectively. Suppression of NF-κB p65 and HIF1α expression significantly limited hypoxia-induced transcriptional activity of both genes (Figure 1e). As expected, suppression of HIF1α inhibited hypoxia-induced BNIP3 expression (Figure 1d). However, suppression of NF-κB p65, but not HIF1α, inhibited hypoxia-induced expression of RAGE protein (Figures 1c and d) and mRNA expression (Figure 1f). Moreover, the NF-κB inhibitor (e.g., ammonium pyrrolidinedithiocarbamate (AP) and Bay 11-7082 (Bay)), but not the HIF1α inhibitor (e.g., methyl 3-((2-(4-(2-adamantyl)phenoxy)acetyl)amino)-4-hydroxybenzoate), inhibited hypoxia-induced RAGE protein (Figure 1g) and mRNA (data not shown) expression. These findings suggest that hypoxia-induced RAGE expression is NF-κB-dependent and HIF1α-independent.

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