YAP AND TEAD ARE TRANSCRIPTIONAL REGULATORS OF NEUROENDOCRINE DIFFERENTIATION AND GROWTH IN CARCINOID CELLS [ChIP-seq]

Molecular regulators of variably aggressive carcinoid tumors are unknown. Since carcinoids have low expression of Yes-associated protein (YAP), we hypothesized that low YAP expression provides a molecular advantage to carcinoids by preventing YAP from binding its partner, TEA domain transcription factor (TEAD). To test this hypothesis, we overexpressed constitutively active YAP and a TEAD-binding defective form of YAP in lung (H727) and pancreatic (BON1) carcinoid cells. We found that active YAP overexpression inhibited neuroendocrine markers, morphology, cell proliferation and anchorage-independent cell growth, while TEAD-binding defective YAP recovered these features. Through integrated ChIP-seq and RNA-seq analyses, we found that YAP-TEAD binding downregulated neuroendocrine transcription factor genes and upregulated transforming growth factor (TGFß) and Notch genes related to cell growth. We conclude that low YAP expression permits neuroendocrine differentiation and growth in carcinoid cells by preventing YAP-TEAD binding and subsequent dysregulation of neuroendocrine transcription factors, TGFß and Notch gene targets. These results identify unknown molecular mechanisms in carcinoid development that may apply to the broader family of neuroendocrine cancers. Overall design: ChIP-seq for YAP and TEAD immunoprecipitation in lung (H727) and pancreatic (BON1) human carcinoid cell lines. Experimental conditions included control and active YAP overexpression.

不同侵袭性类癌肿瘤的分子调控机制目前尚未明确。由于类癌的Yes相关蛋白（Yes-associated protein, YAP）表达水平较低，我们提出假设：低YAP表达可通过阻断YAP与其结合伴侣TEA结构域转录因子（TEA domain transcription factor, TEAD）的结合，赋予类癌分子层面的生长优势。为验证该假说，我们在肺源（H727）与胰腺源（BON1）类癌细胞系中，分别过表达组成型激活的YAP，以及TEAD结合缺陷型YAP。结果显示，活性YAP过表达会抑制神经内分泌标志物的表达、改变细胞形态、阻滞细胞增殖与非锚定依赖性细胞生长；而TEAD结合缺陷型YAP则可逆转上述表型。通过整合染色质免疫共沉淀测序（ChIP-seq）与RNA测序（RNA-seq）分析，我们发现YAP-TEAD结合会下调神经内分泌转录因子基因的表达，并上调与细胞生长相关的转化生长因子β（transforming growth factor-β, TGFβ）与Notch基因的转录水平。综上，低YAP表达可通过阻断YAP-TEAD结合，以及后续对神经内分泌转录因子、TGFβ与Notch基因靶标的异常调控，使类癌细胞得以维持神经内分泌分化状态并实现增殖。本研究阐明了类癌发生过程中此前未知的分子机制，该机制或可推广至更广范围的神经内分泌癌症家族。实验整体设计：在肺源（H727）与胰腺源（BON1）人类类癌细胞系中，针对YAP与TEAD开展染色质免疫共沉淀测序（ChIP-seq）。实验设置包含对照组与活性YAP过表达组。