Endocannabinoid Signaling and Obesity-linked Cancer

用CB1R激动剂Win55,212-2处理PANC-1细胞，可导致配体诱导的EGF受体磷酸化和下游信号转导的时间依赖性减少。相反，AM251抑制CB1R信号转导导致EGF配体(Hereglin和Betacellin)和EGF受体(ErbB1)的表达显著增加。这些结果是通过定量聚合酶链式反应、免疫印迹和EGF/PDGF信号聚合酶链式反应阵列建立的。AM251不增加IGF-1受体的表达，AM630抑制CB2R不影响EGF受体或其配体的表达，表明AM251在这些细胞中的作用具有特异性。流式细胞术分析和生物素探针选择性标记细胞表面蛋白表明，AM251处理的PANC-1细胞质膜上EGF受体表达增加，与EGF单独作用相比，这与EGF依赖的EGF受体及其下游介质Akt的磷酸化水平升高相一致。除了EGF信号的增强外，AM251还引起了明显的细胞形态变化，细胞从立方形状变成具有细长突起的圆形细胞。对AM251的反应中E-钙粘附素的丢失表明这些细胞可能正在经历上皮向间充质(EMT)的转变，这是侵袭性的标志。事实上，根据Matrigel侵袭小室的评估，AM251显著增强了PANC-1细胞的侵袭潜力。目前正在系统地研究多种转录因子的信号通路和活性，这些转录因子直接控制EGF受体及其配体响应AM251的基因表达和功能的程度。这些研究将扩大到包括使用来自其他类型的癌细胞(例如，结肠、乳腺、卵巢)的细胞系和小鼠的肿瘤异种移植模型。

Treatment of PANC-1 cells with the CB1R agonist Win55,212-2 resulted in a time-dependent decrease in ligand-induced phosphorylation of the EGF receptor and downstream signaling. In contrast, inhibition of CB1R signaling by AM251 led to a significant increase in the expression of EGF ligands (heregulin and betacellulin) and EGF receptor (ErbB1). These results were established by quantitative PCR, Western immunoblotting and EGF/PDGF signaling PCR array. There was no increase in IGF-1 receptor expression by AM251, and inhibition of CB2R with AM630 did not affect expression of the EGF receptor or its ligands, indicating the specific nature of AM251 action in these cells. Flow cytometry analyses and selective labeling of cell surface proteins with biotin probe indicated an increase in EGF receptor at the plasma membrane of AM251-treated PANC-1 cells, which coincided with higher levels of EGF-dependent phosphorylation of EGF receptor and its downstream mediator, Akt, when compared to EGF alone. In addition to the potentiation in EGF signaling, AM251 elicited distinct cellular morphological changes, with the cells changing from a cuboidal shape to a rounded cell with elongated projections. The loss of E-cadherin in response to AM251 suggests that these cells may be undergoing an epithelial to mesenchymal (EMT) transition, a hallmark of invasiveness. Indeed, AM251 enhanced significantly the invasive potential of PANC-1 cells as assessed by Matrigel invasion chambers. Work is underway to systematically examine the signaling pathways and activities of multiple transcription factors that directly control the extent of gene expression and function of the EGF receptor and its ligands in response to AM251. These studies will be expanded to include the use of cell lines from other cancer cell types (e.g., colon, breast, ovaries) and tumor xenograft models in mice.