Abstract
We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.
Highlights
Inactivation of the retinoblastoma tumor suppressor protein (Rb) occurs with high frequency as one of the early events in human tumorigenesis [1,2,3,4,5]
We previously showed that Rb-expressing MC3T3 osteoblasts have significantly diminished Pak1 mRNA and protein steady-state levels relative to their Rb-deficient counterparts [16], suggesting that Rb exerts a repressive action on Pak1 expression
Regulation of cell adhesion is one of the latest roles ascribed to Rb, widely regarded as a multifunctional protein
Summary
Inactivation of the retinoblastoma tumor suppressor protein (Rb) occurs with high frequency as one of the early events in human tumorigenesis [1,2,3,4,5]. Pervasive Rb inactivation in an oncogenic context is natural given that Rb has been characterized predominantly as a cell cycle repressor, as the main regulator of the G1-S transition checkpoint [1, 2]. Active Rb binds E2F transcription factors and abrogates their ability to induce S-phase-related gene expression [1, 2]. This diverts cells to G0 rather than allowing their progression to the S phase. Rb inactivation de-regulates the cell cycle and renders cells incapable of exiting a proliferative state
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