Abstract
It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with an overall 5-year relative survival rate below 9% [1]
These results suggest that there is a KRAS mutation-dependent positive correlation in aberrant activation of Hh and nuclear factor-kappa B (NF-kB) pathways in pancreatic ductal adenocarcinoma (PDAC)
To further determine whether crosstalk between Hh and NF-kB pathways is dependent on the presence or absence of KRAS mutation, three different PDAC cell lines (SW1990, Panc-1, and BxPC-3) were chosen based on their specific traits
Summary
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with an overall 5-year relative survival rate below 9% [1]. Based on global genomic analyses, 12 core signaling pathways and processes have been identified as having clear involvement in PDAC carcinogenesis, suggesting that deregulation of these core pathways are involved in the tumorigenesis of pancreatic cancer [3,4,5]. Among these pathways, the Hedgehog (Hh), nuclear factor-kappa B (NFkB), and KRAS signaling pathways have been the most studied and their aberrant activation has been shown to play an important role in the pancreatic carcinogenesis [6, 7]. Shh can induce the activation of the Gli family of transcription factors including Gli, by the classical pathway as well as the nonclassical pathway [15]
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