The chromatin protein Nupr1 regulates RelB-dependent NF-kB events necessary for pancreatic cancer development

The objective of this study was to elucidate the role of Nupr1 in pancreatic tumorigenesis. Using the Pdx-1-cre;LSL-KrasG12D mouse as model we discovered that, in contrast to KrasG12D pancreas that develop multiple foci of pancreatic intraepithelial neoplasia (PanIN), KrasG12D;Nupr1KO pancreas were free from such lesions, indicating that Nupr1 is pivotal for PanIN formation. In vitro, MiaPaCa2 cells activated Nupr1 expression in response to nutrient deprivation and this expression was necessary for cell survival. Mechanistically, Nupr1 protected cells from stress-induced death by inhibiting apoptosis through an alternative RelBàIER3-dependent pathway and independent from activation of the classical RelA-based NF-kB pathway. In agreement with these findings, Nupr1, RelB and IER3 proteins were found co-expressed in PanINs from KrasG12D pancreas. Moreover, pancreas-specific KrasG12D;RelbDpanc mice displayed a delay in PanIN development associated with a lack of IER3 expression, further emphasizing the relevance of this pathway in vivo. Efficient PanIN formation was therefore dependent on the expression of Nupr1 and RelB, with the probable involvement of IER3. Finally, a significant correlation between expression of Nupr1, RelB and IER3 and a poor prognosis of patients with PDAC was found. Altogether, our results reveal a novel stress-related pathway that requires the functional interaction of Nupr1àRelBàIER3 in KrasG12D-dependent transformation of the pancreas and expand our understanding of the molecular machinery that mediates the early steps of pancreatic carcinogenesis. Since Nupr1 belongs to the HMG family of chromatin remodelers with transcriptional co-factor activity, Nupr1 could increase cell survival in a nutrient-deprived microenvironment by activating the expression of pro-survival genes. Moreover, considering that RelB, and not RelA/p65, is essential to the Nupr1-mediated survival mechanism that takes place upon nutrient deprivation-induced stress, we made the hypothesis that the two NF-kB transcription factors should activate different sets of genes among which a pivotal pro-survival gene would be exclusively dependent on RelB. In order to test this two hypothesis, an Affymetrix microarray analysis was performed using pancreatic cancer cells transfected with siCtrl or siNupr1 and cultured for 3, 6 or 9 hrs in EBSS; or transfected with siRelB, siRelA/p65 or siCtrl and cultured for 9 hrs in EBSS or Mock.

本研究旨在阐明核蛋白1（Nupr1）在胰腺肿瘤发生中的作用。以Pdx-1-cre;LSL-KrasG12D小鼠为模型，我们发现：与形成多处胰腺上皮内瘤变（pancreatic intraepithelial neoplasia，PanIN）病灶的KrasG12D小鼠胰腺不同，KrasG12D;Nupr1KO小鼠胰腺未出现此类病变，提示Nupr1对PanIN形成至关重要。体外实验中，MiaPaCa2细胞在营养匮乏条件下会激活Nupr1的表达，且该表达对细胞存活不可或缺。机制层面，Nupr1通过一条依赖于RelB→IER3的非经典通路抑制细胞凋亡，从而保护细胞免受应激诱导的死亡，且该过程不依赖经典的RelA介导的核因子κB（NF-κB）通路激活。与上述发现一致，我们在KrasG12D小鼠胰腺的PanIN病灶中检测到Nupr1、RelB及IER3蛋白共表达。此外，胰腺特异性敲除Relb的KrasG12D;RelbΔpanc小鼠的PanIN发育进程延迟，且未检测到IER3表达，进一步验证了该通路在体内的生物学意义。因此，有效的PanIN形成依赖于Nupr1与RelB的表达，且可能涉及IER3的参与。最后，我们发现胰腺导管腺癌（PDAC）患者中，Nupr1、RelB与IER3的表达水平与不良预后显著相关。综上，我们的研究揭示了一条全新的应激相关通路，该通路需要Nupr1→RelB→IER3的功能协同参与胰腺KrasG12D介导的转化过程，加深了我们对胰腺癌变早期分子机制的理解。鉴于Nupr1属于具有转录辅因子活性的高迁移率族（HMG）家族染色质重塑因子，其可通过激活促存活基因的表达，在营养匮乏的微环境中提升细胞存活能力。此外，考虑到介导营养匮乏应激下细胞存活的Nupr1信号通路依赖RelB而非RelA/p65，我们提出假说：这两种NF-κB转录因子可调控不同的基因集，其中关键的促存活基因仅由RelB介导激活。为验证该假说，我们采用Affymetrix基因芯片技术，对分别转染siCtrl或siNupr1、并在Earle平衡盐溶液（EBSS）中培养3、6或9小时的胰腺癌细胞进行分析；同时也对转染siRelB、siRelA/p65或siCtrl、并在EBSS或模拟转染组（Mock）中培养9小时的细胞进行了检测。