The members of the retinoblastoma (RB) protein family, RB1/p105, retinoblastoma-like (RBL)1/p107 and RBL2/p130 are critical modulators of the cell cycle and their dysregulation has been associated with tumor initiation and progression. The activity of RB proteins is regulated by numerous pathways including oncogenic signaling, but the molecular mechanisms of these functional interactions are not fully defined. We previously demonstrated that RBL2/p130 is a direct target of AKT and it is a key mediator of the apoptotic process induced by AKT inhibition. Here we demonstrated that RBL1/p107 levels are only minorly modulated by the AKT signaling pathway. In contrast, we discovered that RBL1/p107 levels are regulated by multiple pathways linked directly or indirectly to Ca2+-dependent signaling. Inhibition of the multifunctional calcium/calmodulin-dependent kinases (CaMKs) significantly reduced RBL1/p107 expression levels and phosphorylation, increased RBL1/p107 nuclear localization and led to cell cycle arrest in G0/G1. Targeting the Ca2+-dependent endopeptidase calpain stabilized RBL1/p107 levels and counteracted the reduction of RBL1/p107 levels associated with CaMKs inhibition. Thus, these novel observations suggest a complex regulation of RBL1/p107 expression involving different components of signaling pathways controlled by Ca2+ levels, including CaMKs and calpain, pointing out a significant difference with the mechanisms modulating the close family member RBL2/p130.

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107, and RBL2/p130, coordinately represses cell cycle gene expression, inhibiting proliferation, and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin-dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.