Novel noncanonical actions of CAR in human Liver

项目摘要 结构性雄烷受体(CAR；NR1I3)是一种成熟的异种生物传感器，调节 大量编码对药物代谢和清除重要的蛋白质的基因的表达。积累 有证据表明，CAR在协调不同的生理和生理功能方面也发挥着非典型的作用 与能量平衡和细胞增殖相关的病理生理反应。对啮齿动物的研究已经 证实CAR的激活是促进肝肿瘤形成的关键事件。相比之下，汽车激活- 两种培养方式均未观察到啮齿动物复制DNA合成和肝细胞增殖。 人肝细胞在体外或与人源化肝脏嵌合的小鼠体内。此外，流行病学研究已经 研究表明，即使在长期临床使用后，典型的CAR激活剂苯巴比妥也不会增加 人类肝脏肿瘤的发病率。然而，人CAR(Hcar)在肝癌细胞增殖和肝脏中的作用 癌症的发展仍然知之甚少。这个应用程序的总体目标是描述角色 HCAR在肝肿瘤进展中的作用及对其分子机制的全面认识 HCAR对肝癌细胞增殖的影响。为此，我们已经证明了1)表达式 肝细胞癌(HCC)的HCAR显著低于正常肝脏，更重要的是， Hcar的表达与肝细胞癌的预后呈负相关；2)参考Hcar的异位表达 非剪接变异体(HCAR3)在肝癌细胞中明显抑制细胞增殖、软琼脂集落形成 裸鼠移植瘤的形成和生长；3)rna-seq分析显示hcar改变。 包括促红细胞生成素下调在内的一系列抑癌基因和癌基因的表达 促红细胞生成素(EPO)，一种具有细胞增殖和抗凋亡功能的多效性生长因子；以及4)激活 人和小鼠CAR在体内差异地改变细胞增殖基因的表达。基于这些 令人兴奋的初步发现，我们假设与其啮齿动物同行形成鲜明对比的是，Hcar展示了 抗癌作用是通过下调EPO来抑制肝细胞癌的进展。这一中心假说将 目的1.明确HCAR亚型在肝癌细胞增殖和肝细胞癌中的作用 目的2.描述HCAR抑制肝细胞癌进展的机制。我们的发现 有望确定Hcar在肝癌发展中的作用，并提供新的机制见解 HCAR介导的对肝癌进展的抑制将为新的生物标记物和治疗方法打开大门。

Project Summary The constitutive androstane receptor (CAR; NR1i3) is a well-established xenobiotic sensor that regulates the expression of numerous genes encoding proteins important for drug metabolism and clearance. Accumulating evidence suggests that CAR also plays noncanonical roles in coordinating diverse physiological and pathophysiological responses associated with energy homeostasis and cell proliferation. Studies in rodents have established activation of CAR as a key event promoting liver tumor formation. In contrast, CAR activation- induced replicative DNA synthesis and hepatocyte proliferation in rodents were not observed in either cultured human liver cells in vitro or in chimeric mice with humanized liver in vivo. Moreover, epidemiological studies have shown that even after long-term clinical use, phenobarbital, a prototypical CAR activator, does not increase the incidence of liver tumors in humans. Yet, the role of human CAR (hCAR) in hepatoma cell proliferation and liver cancer development remains poorly understood. The overall objective of this application is to delineate the role of hCAR in liver tumor progression and to develop a comprehensive understanding of the molecular mechanisms underlying the effects of hCAR on hepatoma cell proliferation. To this end, we have shown that 1) expression of hCAR was significantly lower in hepatocellular carcinoma (HCC) compared to normal liver and, importantly, hCAR expression is inversely correlated with HCC outcomes; 2) ectopic expression of the reference hCAR but not a splicing variant isoform (hCAR3) in hepatoma cells markedly repressed cell proliferation, soft agar colony formation, and the growth of hepatoma xenografts in nude mice; 3) RNA-seq analyses revealed that hCAR alters the expression of a cluster of tumor suppressors and oncogenes including the downregulation of erythropoietin (EPO), a pleiotropic growth factor that exhibits cell proliferation and anti-apoptosis functions; and 4) activation of human and mouse CAR differentially alters the expression of cell proliferation genes in vivo. Based on these exciting preliminary findings, we hypothesize that in stark contrast to its rodent counterparts, hCAR exhibits anticancer functions that repress the progression of HCC by downregulating EPO. This central hypothesis will be tested in two Specific Aims: Aim 1. Define the role of hCAR isoforms in hepatoma cell proliferation and HCC progression; and Aim 2. Delineate the mechanisms by which hCAR represses HCC progression. Our findings are expected to determine the role of hCAR in HCC development and provide novel mechanistic insights into hCAR-mediated suppression of HCC progression that will open the door to novel biomarkers and therapeutics.