Wang Yuqiong, Wang Dan, Dai Yanmiao, Kong Xiangyu, Zhu Xian, Fan Yunxia, Wang Yaodong, Wu Hongyu, Jin Jing, Yao Wenzhu, Gao Jun, Wang Kaixuan, Xu Hongwei
Department of Gastroenterology, the Hospital of 92608 People's Liberation Army of China (PLA) Troops, Shanghai, China.
Institute of Oncology, Second Affiliated Hospital, Xi'an Medical University, Xi'an, China.
Front Oncol. 2021 May 11;11:652283. doi: 10.3389/fonc.2021.652283. eCollection 2021.
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 as well as tumor growth . 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 and tumor growth after inoculation with MT-KRAS cells 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.
研究表明，刺猬信号通路（Hh）和核因子-κB（NF-κB）信号通路的异常激活在胰腺癌发生过程中起重要作用，而KRAS突变是胰腺导管腺癌（PDAC）的一个标志。到目前为止，KRAS突变在PDAC中Hh和NF-κB信号通路相互作用背景下的作用尚未得到研究。本研究旨在确定PDAC中Hh和NF-κB通路之间的相互作用是否依赖于KRAS突变。首先通过免疫组织化学检测PDAC组织中Gli1、Shh、NF-κB p65表达与KRAS突变之间的相关性。接下来，进行蛋白质免疫印迹、定量聚合酶链反应和免疫荧光检测，以研究白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）作为NF-κB信号激动剂、Shh作为单独的Hh配体或与KRAS小干扰RNA（si-KRAS）联合使用时，对KRAS突变的PDAC细胞（MT-KRAS；SW1990和Panc-1）、野生型KRAS的PDAC细胞（WT-KRAS；BxPC-3）和KRAS突变敲入的BxPC-3细胞以及肿瘤生长的生物学效应。通过Ras活性和荧光素酶报告基因检测进一步评估PDAC细胞中Hh和NF-κB之间KRAS突变依赖性的相互作用。在有KRAS突变的PDAC组织中发现了Hh和NF-κB通路的异常激活。在MT-KRAS PDAC细胞和MT-KRAS敲入的BxPC-3细胞中也证实了相同的结果，而在WT-KRAS PDAC细胞中未观察到这种激活。然而，在MT-KRAS PDAC细胞中，KRAS沉默可显著下调这种激活。此外，NF-κB和Hh信号激活促进了MT-KRAS癌细胞的增殖、存活以及接种MT-KRAS细胞后的肿瘤生长。NF-κB和Hh信号共同激活的关键因素是MT-KRAS蛋白上调，表明Hh和NF-κB通路之间的正向相互作用依赖于PDAC中的KRAS突变。
