Beta-arrestins in cancer : linking pro-tumorigenic extracellular activated signaling with the tumor suppressor p53 pathway

Sammanfattning: The IGF-1R is an important player in cancer development that maintains the malignant phenotype by inducing cell proliferation, survival, transformation, motility and invasiveness. Activation of IGF-R results in its Mdm2-dependent ubiquitination and degradation followed by MAPK signaling. IGF-1R ubiquitination by Mdm2 is mediated by scaffolding protein β- arrestin 1. The tumor suppressor p53 pathway is activated in damaged cells causing growth arrest and if necessary, apoptosis and senescence. In normal conditions p53 is inactivated by Mdm2. Activation of extracellular pro-survival signaling has been shown to inhibit p53 activity through β-arrestin 1. Thus the same Mdm2/β-arrestin 1 system regulates two important pathways involved in cancer. The aim of this thesis was to investigate in detail the IGF-1R/β-arrestin/Mdm2/p53 axis and explore the potential use of its components as anti-cancer therapeutic targets. In Paper I we analysed the molecular interplay between p53 and IGF-1R through Mdm2. We tested the effect of p53/Mdm2 disruption on IGF-1R using a panel of melanoma cells and the p53-rectivator Nutlin-3. Disruption of the p53/Mdm2 interaction by Nutlin-3 increased the IGF-1R/Mdm2 interaction, followed by IGF-1R degradation and MAPK activation. This resulted in reduced cell proliferation and invasion and had a two-step effect on cell migration, demonstrating that modulation of the p53/Mdm2/IGF-1R axis is a potential anti-cancer therapeutic strategy. In paper II we focused on the role of β-arrestin isoforms in the p53/Mdm2/IGF-1R axis. By modulating levels of β-arrestin 1 or 2 we identified opposing roles of isoforms on IGF-1R degradation, signaling and p53 pathway. We revealed a higher affinity of ligand-free IGF-1R for β-arrestin 2, and ligand occupied receptor - for β-arrestin 1. Antagonism between isoforms was also observed on biological effects with β-arrestin 2 causing cell cycle arrest and inhibiting IGF-1 response and cell viability, and β-arrestin 1 acting in the opposite direction. Thus we identified the β-arrestin 1/2 system as a second potential drug target within the p53/Mdm2/IGF-1R axis. In paper III we studied the possibility of co-targeting the p53/Mdm2/IGF-1R and the MAPK pathway in melanoma cell lines. We combined MEK inhibitors with 1) balanced IGF-1R inhibition by siRNA; 2) biased IGF-1R inhibition by Nutlin-3, inducing transient MAPK; and 3) biased IGF-1R inhibition by antibody CP, inducing prolonged MAPK. We identified strong synergy between Nutlin-3 and MEK inhibitors. This combination of specific biased IGF-1R inhibition with MEK inhibitors is the first rational anti-cancer strategy identified in this thesis. In paper IV we investigated the possibility of co-targeting the β-arrestin system with the DNA-damage inducing drug dacarbazine. By modulating the level of β-arrestin isoforms in melanoma cell lines we demonstrated that both β-arrestin 1 inhibition and β-arrestin 2 overexpression synergize with dacarbazine. This study revealed the second rational anticancer strategy of this project. To sum up, our findings demonstrate that the p53/Mdm2/IGF-1R axis is a potential target for anti-cancer therapy. However, optimal effects can be achieved only through accurate modulation of multiple pathways regulated by the axis.