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.
| Fiori, J L; Sanghvi, M; O'Connell, M P et al. (2011) The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor ? protein. Br J Pharmacol 164:1026-40 |
| Gandhari, Mukesh K; Frazier, Chester R; Hartenstein, Julia S et al. (2010) Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells. Mol Cell Endocrinol 315:314-8 |
