The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

• Published in: PloS one Vol. 10; no. 11; p. e0142406
• Main Authors: Sosa-García, Bernadette, Vázquez-Rivera, Viviana, González-Flores, Jonathan N, Engel, Brienne E, Cress, W Douglas, Santiago-Cardona, Pedro G
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.11.2015; Public Library of Science (PLoS)
• Subjects: 3T3 Cells; Adapter proteins; Adenoviruses; Adhesion; Animals; Assembly; Base pairs; Binding sites; Biochemistry; Biocompatibility; Bioinformatics; Bone cancer; Breast cancer; Cancer; Cell adhesion; Cell adhesion & migration; Cell cycle; Cell migration; Chromatin; Cytogenetics; Cytoskeleton; Development and progression; E2F1 protein; E2F1 Transcription Factor - metabolism; Etiology; Gene expression; Gene Silencing; GTP-binding protein; Guanosine triphosphatases; Hyperactivity; Kinases; Labeling; Luciferase; Lung cancer; Medical research; Metastases; Metastasis; Mice; Molecular modelling; mRNA stability; Oncology; Osteoblasts; Osteoblasts - cytology; Osteoblasts - metabolism; p21-Activated Kinases - genetics; p21-Activated Kinases - metabolism; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein kinase; Rac1 protein; Regulators; Retina; Retinoblastoma; Retinoblastoma protein; Retinoblastoma Protein - metabolism; Retinoblastoma Protein - physiology; RNA; Sarcoma; Science; Solid tumors; Stem cells; Transcription (Genetics); Transcription, Genetic - physiology; Tumor suppressor genes; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0142406

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.