Dissecting the mechanism in blocking tumorigenesis following loss of RB1

Abstract

Retinoblastoma (RB1) is a tumor suppressor protein that is functionally inactivated or deleted in many cancers. RB1+/- mice develop pituitary and thyroid tumors after loss of heterozygosity. Murine inactivation of RB1 downstream targets (such as E2F family members) on the RB1 null background delays tumorigenesis. However inactivation of the F-box protein Skp2, which targets several cyclin dependent kinase inhibitors for ubiquitination, blocks tumorigenesis when RB1 is deleted. In this study, we utilized various mouse models to determine the functions of Skp2 that contribute RB1 deficient tumorigenesis.;Artificial deletion of RB1 in the IL induces tumorigenesis but inactivation of Skp2 in RB1-deleted intermediate lobes blocks tumorigenesis. Interestingly, RB1-/-; Skp2-/- ILs block tumorigenesis and increase apoptosis, indicating that RB1 loss and Skp2 loss have a synthetic lethal relationship.;To assess if the effects of Skp2 loss are due to ubiquitination of p27 we bred mice with the p27T187A mutation that stabilizes p27 onto the RB1 null background. RB1-/-; p27T187A/T187Aphenocopies the effects of RB1-/-; Skp2-/- ILs, indicating that RB1 loss and the p27 T187A KI mutation also have a synthetic lethal relationship.;To further elucidate the molecular pathways involved in this synthetic lethal phenotype, we created RB-/-; E2F1-/- mice. These mice show normal IL architecture and rescued the aberrant proliferation seen in RB-/- ILs. Additionally, inactivation of E2FI on the RB-/-; p27T187A/T187A background also shows normal IL architecture, indicating that deregulated E2F1 is a required for aberrant proliferation and synthetic lethality.;To determine if the phenotypes observed by disrupting the RB1-Skp2-p27 pathway are p53 dependent, Rb-/-p53-/- mice were bred and displayed accelerated tumorigenesis with a loss of normal IL architecture. The p27T187A KI mutation blocks tumorigenesis in Rb-/-p53-/- mice and restores the normal architecture but the ILs are thickened. Similar results are seen when Skp2 is inactivated on the same background. These results indicate that stabilization of p27 blocks RB1-deficient tumorigenesis in a p53 dependent and independent manner.;Together these results dissect the functions of Skp2 that lead to the synthetic relationship between RB1 loss and Skp2 loss.