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.