关于Kras驱动胰腺癌的转录决定因素的探究

Pancreatic ductal adenocarcinoma is a uniformly fatal malignancy that currently has no effective therapy. PDA is initiated by mutations in the KRAS gene, which is altered in approximately 90% of all PDA cases. In addition to KRAS mutations, PDA has an additional driver, pancreatitis, which is an inflammatory condition of the pancreas that is a major risk factor for the development of PDA. Mouse models have shown that Kras mutations cooperate with inflammation to result in the appearance of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDA. The molecular basis of this cooperation is currently unclear. Our previous work showed that PDA tumors depend on a set of highly expressed lineage-specific transcription factors that include Sox4 and Klf5 for growth and survival. Importantly, the emergence of Klf5 expression appears to be driven by the process of inflammation. These observations point to a potential explanation for the close links between inflammation and Kras-driven tumorigenesis. We hypothesize that Kras depends on Klf5, and potentially other TFs that emerge in the inflamed pancreatic epithelium to establish its tumorigenic program of gene expression. To address this possibility, we will use a series of genetically engineered animals to examine the relationship between the expression of key transcription factors and Kras-driven tumorigenesis. First, we will use single cell RNA-seq in a mutant Kras-driven model of pancreatic cancer to examine the emergence of neoplastic cells after the induction of inflammatory pancreatitis. Then, using a Klf5 mCherry-CreERT2 knockin mouse and lineage tracing, we will examine whether Klf5-expressing cells that emerge during inflammatory pancreatitis can serve as a cell of origin for Kras-driven neoplasia. Using the same knockin mouse, we will examine the possibility that Klf5-expressing cells in established tumors possess stem-like properties. Using a Tet-On KrasG12D mouse model, we will examine how different cell types in the pancreas respond to Kras expression, with the hypothesis that only Klf5-expressing cells that emerge during inflammation are capable of activating a tumorigenic gene expression program in response to mutant Kras expression. The proposed experiments have the potential to contribute important molecular insights into the process of Kras-driven tumorigenesis.

胰腺导管腺癌（PDA）是类高致死且无有效治疗方法的肿瘤。其中约90%由KRAS突变引起。除突变外，炎症也是主要风险因子。转基因小鼠揭示Kras突变协同炎症致胰腺上皮内瘤变。但其协同机制尚不明。我已揭示PDA的生长生存依赖Sox4和Klf5等高表达的谱系特异性转录因子。更重要的是，炎症驱动Klf5表达，使其与KRAS驱动癌化紧密联系起来。Kras可能依靠Klf5等出现在炎性胰腺上皮内的转录因子建立癌化基因表达谱。为验证该推论，我们首先对Kras突变小鼠因胰腺炎诱导的病灶前体单细胞测序。再通过Klf5-mCherry-CreERT2小鼠追溯PDA的细胞起源。之后，通过Kras突变Tet-On可调控小鼠检测各类型细胞对Kras激活的反应，尤其是Klf5阳性细胞是否激活Kras突变所致的癌化基因表达谱。本研究对洞悉Kras驱动癌化过程中的重要分子具有深远的意义。

Pancreatic Intraepithelial Neoplasia(PanIN)作为pancreatic ductal adenocarcinoma （PDAC）发展过程中的一个阶段，常常被视为胰腺癌的前体细胞。然而PanIN向PDAC发展的推动力却很少被报道。..我们发现，相较于PanIN细胞，PDAC细胞中大量与“细胞侵袭”相关的基因通路处于转录激活的状态。同时相较于PanIN细胞，PDAC细胞中大量的染色质处于开放程度更高的状态，染色质基序分析还发现AP1转录因子家族的基序被富集到这些染色质位置。之后，我们发现，相较于PanIN细胞，AP1转录因子家族的Junb与Fosl1在PDAC细胞中有更高的表达。在小鼠胰腺中表达Fosl1蛋白能够帮助胰腺突破PanIN细胞的限制，发展为侵袭性更强的PDAC样细胞。除此之外，在胰腺癌的发展过程中，出现了一群即表达AP1转录因子，又表达肿瘤抑癌基因的细胞群。我们利用Cdkn2a-d2eGFP KI细胞系模拟了这一体内的细胞群，发现这群高表达AP1转录因子与Cdkn2a的细胞，拥有更开放的PDAC相关的染色质区域。