Metastatic site influences driver gene function in pancreatic cancer [InVitro RNA-Seq]

Driver gene mutations can increase the metastatic potential of the primary tumor, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 – a transcriptional effector of TGFß signaling – which is a hallmark of multiple gastrointestinal malignancies. SMAD4 inactivation mediates TGFß's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene–chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease. Overall design: SMAD4 acts as a transcription factor (TF) by forming a complex with the SMAD2/3 TFs to activate gene expression programs downstream of TGFß receptors. Hence, we performed RNA-seq of the input cells in vitro after Smad4 restoration and TGFb treatment (see Methods), and compared the resulting pathway enrichment to our in vivo data from the pancreas, liver and lungs.

驱动基因突变可增强原发肿瘤的转移潜能，但此类突变在转移灶维持肿瘤生长过程中所发挥的作用仍有待阐明。此类突变的典型代表为SMAD4——转化生长因子β (TGF-β) 信号通路的转录效应因子，该突变是多种胃肠道恶性肿瘤的标志性特征。SMAD4失活可介导TGF-β在癌症进展过程中从抗肿瘤向促肿瘤发生的关键转变，因此可同时影响肿瘤起始与转移过程。为明确转移性肿瘤是否仍依赖SMAD4失活，我们构建了一款胰腺导管腺癌 (PDAC) 小鼠模型，该模型可在癌前胰腺组织中实现Smad4敲除，并可在已形成的转移灶中重新激活Smad4的表达。如预期所示，Smad4失活可促进原发肿瘤的形成，并最终使肿瘤定植于肝脏与肺部。与之相反，转移灶中的Smad4重新激活则会根据肿瘤所处的器官微环境产生截然相反的效应：抑制肝脏转移瘤的生长，同时促进肺部转移瘤的增殖。整合多组学分析结果显示，肿瘤细胞的表观基因组状态存在器官特异性差异：肝脏与肺部转移瘤的染色质调控程序分别由KLF家族与RUNX家族发育转录因子主导，且敲低Klf4即可逆转Smad4在肝脏转移瘤中的抗肿瘤活性。本研究结果阐明了器官微环境所偏好的表观基因组状态，如何调控驱动基因在转移性肿瘤中的功能。这种器官特异性的基因-染色质互作机制提示，在解读肿瘤遗传学特征时需纳入解剖部位的考量，这对转移性肿瘤的靶向治疗具有重要指导意义。实验整体设计：SMAD4可与SMAD2/3转录因子 (TF) 形成复合物，作为转录因子激活TGF-β受体下游的基因表达程序。因此，我们在体外对经Smad4恢复表达及TGF-β处理的输入细胞进行了RNA测序（详见方法部分），并将所得的通路富集结果与我们在胰腺、肝脏及肺部获取的体内数据进行了对比。