Investigating molecular regulators of a mixed-lineage state in lung adenocarcinoma

项目摘要： 肺腺癌（LUAD）是肺癌中最大的亚型，也是最常见的癌症病因 相关死亡LUAD进展由遗传改变和表观遗传变化的组合驱动， 赋予增加的可塑性和异质性，但潜在的机制决定细胞的命运和可塑性， 对LUAD的了解仍然很少。有趣的是，谱系定义转录因子NKX 2 -1和FoxA 1/2， 协调地维持LUAD中的肺部特征。在NKX 2 -1缺乏的情况下，FoxA 1/2可以激活胃粘膜， 包括HNF 4 α在内的转录因子，以显著改变LUAD的分化状态。最近的工作 提示NKX 2 -1阳性（NKX 2 -1+）LUAD从肺泡样状态演变而来，并采用各种不同的 细胞命运驱动进展，包括“混合谱系”状态，其特征在于获得胃肠道 (GI)和类肺动脉疾病。这项建议将调查这些胃的国家是如何 维持以及它们如何促进LUAD肿瘤发生。HNF 4 α水平在LUAD过程中增加 HNF 4 α和NKX 2 -1在人和小鼠NKX 2 -1+ LUAD的亚组中共表达 肿瘤的这支持了HNF 4 α驱动LUAD进展并促进混合谱系的假定模型。 通过与NKX 2 -1竞争FoxA 1/2结合来表达。此外，鉴于先前的发现，MAPK信号转导， 对于LUAD进展和肺泡命运是重要，已知NKX 2 -1被磷酸化， MEK/ERK信号转导被ERK失活，可能通过使NKX 2 -1/FoxA 1/2失稳而促进混合谱系状态 相互作用和有利于HNF 4 α/FoxA 1/2结合。我推测HNF 4 α促进LUAD肿瘤发生 和增殖，MAPK信号转导通过改变活性调节混合谱系状态， 关键世系说明符的相互作用。为了验证这一假设，我将使用 互补的药理学和遗传学方法并评估关键混合谱系基因的表达， 以及使用邻近连接测定在MAPK操作后显现蛋白质复合物。此外，本发明还 我将进行ChIP-seq，以确定调节MEK/ERK如何改变NKX 2 -1和FoxA 1/2染色质结合 胃和肺的靶点我还将扩展我们小组的初步数据，表明HNF 4 α 在鼠类器官以及KRAS驱动的LUAD小鼠模型中驱动肿瘤发生和增殖。我会 在表达NKX 2 -1和HNF 4A的人LUAD细胞系中遗传调节HNF 4A， 增殖测定，并创建细胞系异种移植模型以测量肿瘤生长。我将执行综合 RNA-seq和ChIP-seq分析，以了解HNF 4 α介导的生长和进展的关键因素 在NKX 2 -1+ LUAD中。这些目标的完成将揭示对LUAD机制的关键见解， 异质性驱动肿瘤生长和进展，这对于开发有效的， 针对这种疾病的谱系靶向疗法。这项提案还将探讨创新的概念，即致癌 信号传导途径和关键转录因子协同调节癌症中的细胞命运。

PROJECT ABSTRACT: Lung adenocarcinoma (LUAD) is the largest subtype of lung cancer and is the most common cause of cancer related death. LUAD progression is driven by a combination of genetic alterations and epigenetic changes that confer increased plasticity and heterogeneity, but underlying mechanisms dictating cell fate and plasticity in LUAD remain poorly understood. Interestingly, the lineage defining transcription factors, NKX2-1 and FoxA1/2, coordinately maintain a pulmonary identity in LUAD. In the absence of NKX2-1, FoxA1/2 can activate gastric transcription factors including HNF4α to substantially alter the differentiation state of LUAD. Recent work suggests that NKX2-1-positive (NKX2-1+) LUAD evolves from an alveolar-like state and adopts various disparate cell fates that drive progression, including “mixed lineage” states, characterized by acquisition of gastrointestinal (GI) and pulmonary-like states in the same cell. This proposal will investigate how these gastric states are maintained and how they may promote LUAD tumorigenesis. HNF4α levels increase over the course of LUAD progression, and HNF4α and NKX2-1 are co-expressed in a subset of human and mouse NKX2-1+ LUAD tumors. This supports a putative model by which HNF4α drives LUAD progression and promotes a mixed-lineage state by competing with NKX2-1 for FoxA1/2 binding. Additionally, given previous findings that MAPK signaling is important for LUAD progression and alveolar fate, and that NKX2-1 is known to be phosphorylated and inactivated by ERK, MEK/ERK signaling may promote mixed-lineage states by destabilizing NKX2-1/FoxA1/2 interactions and favoring HNF4α/FoxA1/2 binding. I hypothesize that HNF4α promotes LUAD tumorigenesis and proliferation, and that MAPK signaling modulates the mixed-lineage state by altering the activity and interactions of key lineage specifiers. To test this hypothesis, I will modulate MAPK signaling using complementary pharmacological and genetic approaches and assess expression of key mixed lineage genes, as well as visualizing protein complexes upon MAPK manipulation using a Proximity Ligation Assay. Additionally, I will perform ChIP-seq to determine how modulating MEK/ERK alters NKX2-1 and FoxA1/2 chromatin binding at gastric and pulmonary targets. I will also expand on preliminary data from our group demonstrating that HNF4α drives tumorigenesis and proliferation in murine organoids as well as a KRAS-driven LUAD mouse model. I will genetically modulate HNF4A in human LUAD cell lines expressing both NKX2-1 and HNF4A, perform proliferation assays, and create cell line xenograft models to measure tumor growth. I will perform integrative RNA-seq and ChIP-seq analysis to understand key factors underlying HNF4α-mediated growth and progression in NKX2-1+ LUAD. Completion of these aims will unveil key insights into the mechanisms by which LUAD heterogeneity drives tumor growth and progression, knowledge which will be critical for development of effective, lineage-targeted therapies for this disease. This proposal will also explore the innovative notion that oncogenic signaling pathways and key transcription factors coordinately regulate cellular fate in cancer.