Regulated expression of the orphan nuclear estrogen-related receptor alpha

早期的研究已经描述了AM251在野生型和CB1R敲除小鼠中不依赖CB1R的作用。在此，我们观察到AM251在人PANC-1胰腺癌细胞系和HCT116结肠癌细胞中对EGFR及其配体表达的有效和协调诱导。这一事件与细胞表面EGFR的表达增强有关，同时在am251处理的细胞中，egf诱导的细胞反应也随之增加。利用遗传、生化和药理学方法，我们发现这种转录反应与大麻素1受体的调节无关，而是依赖于AM251s作为雌激素相关受体的逆激动剂的作用[#945；]。ERR&#945；是一种孤儿核受体，其在肿瘤细胞中的表达与不良预后相关。暴露于合成雌激素己烯雌酚或经典逆转录激动剂XCT790也能在转录水平上诱导EGFR和配体，其程度与AM251相同，而使用err&# 945；选择性激动剂生物茶素A预处理会减弱AM251的作用。AM251促进err&# 945蛋白的蛋白水解降解，而不丢失相应的mRNA。通过基于sirna的方法敲低ERR&#945，导致EGFR及其配体的组成性诱导，并消除AM251和XCT790的生物学反应。最后，发现AM251取代了预结合到ERR&#945；配体结合域的己烯雌酚。综上所述，本研究表明CB1R逆激动剂AM251在培养的人类癌细胞系中通过结合孤儿核受体ERR&#945；并使其失稳而发挥脱靶效应。由于AM251及其临床类似物利莫那班的结构特征，未来的研究将需要在正常细胞系和癌细胞系以及体内模型中进行，以确定这些化合物作为潜在的内分泌干扰化学物质和致癌剂的特性。

Earlier studies have described CB1R-independent actions of AM251 in wild-type and CB1R knockout mice. Here we observed a potent and coordinate induction in the expression of EGFR and its ligands by AM251 in the human PANC-1 pancreatic cancer cell line and HCT116 colon carcinoma cells. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Using genetic, biochemical and pharmacological approaches, we showed that this transcriptional response was NOT related to the modulation of cannabinoid 1 receptor, but, instead, relied on AM251s role as an inverse agonist of estrogen-related receptor α (ERRα). ERRα is an orphan nuclear receptor, whose expression in tumor cells is associated with bad prognosis. Exposure to the synthetic estrogen diethylstilbestrol or the classical inverse agonist XCT790 also induced EGFR and ligands at the transcriptional level to the same extent as AM251, whereas pretreatment with the ERRα selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the proteolytic degradation of ERRα protein without loss of the corresponding mRNA. Knockdown of ERRα by siRNA-based approach led to constitutive induction of EGFR and its ligands and eliminated the biological responses of AM251 and XCT790. Finally, AM251 was found to displace diethylstilbestrol prebound to the ligand-binding domain of ERRα. In summary, this study demonstrates that the CB1R inverse agonist, AM251, exerts off-target effects through binding to and destabilization of the orphan nuclear receptor ERRα in cultured human cancer cell lines. Because of the structural characteristics of AM251 and its clinical analog, rimonabant, future studies will need to be carried out in a panel of normal and cancer cell lines and in vivo models to determine the properties of these compounds as potential endocrine-disrupting chemicals and cancer-inducing agents. Work is underway to determine whether pharmacological interventions provide a viable avenue for interfering with ERRα protein stability in cellular models of various human cancer types and mouse xenograft models. Because of the newly discovered role for ERRα in adaptive energy metabolism, strategies aimed at targeting ERRα may be useful in fighting not only cancer but also metabolic diseases as well. A new investigation on the effect of AM251 on ERRα transcriptional activity will be initiated soon, focusing on histone acetylation and DNA methylation, two major epigenetic molecular mechanisms involved in activation or repression of transcription through changes in chromatin configurations. It is likely that the recruitment of nuclear co-regulatory molecules and other DNA-binding proteins to ERRα at the target gene promoter region is mediated by a mechanism involving epigenetic alterations.