Abstract 206: Molecular mechanisms of aggressiveness of osteosarcomas.
Authors: Engel Brienne , Santiago-Cardona Pedro G. , Sosa-García Bernadete , Cress Douglas 
Published: August 2014

Abstract
Osteosarcomas (OS) are highly aggressive neoplasms of osteoblastic origin. Evidencing their early proclivity to metastasize, at the time of diagnosis most OS are already classified as high grade and poorly differentiated malignancies and about 20% have detectable metastases. Prognosis is very poor with only about 10% of patients achieving long-term disease-free intervals. In spite of this, OS aggressivity still remains to be mechanistically explained. Our work sheds light into this issue by uncovering a novel function for the retinoblastoma protein (Rb), a cell cycle repressor mutated in most OS, in promoting cell adhesion. Our microarrays comparing Rb-expressing with Rb-deficient osteoblasts showed that Rb regulates a wide variety of cell adhesion genes, specifically up-regulating the expression of osteoblast -specific cadherins and integrins, while down-regulating the expression of several metastasis-associated cell adhesion molecules. Cellular pathway analyses showed that 8 of the top 10 cellular processes affected by Rb are related to cell adhesion, with integrin- and adherens junctions-mediated adhesion having the first and fourth best p-values in the list, respectively. This regulation was confirmed when we re-introduced Rb into the Rb-null OS cell line Saos-2 and found an Rb-engendered change in the expression profile of several cadherins and integrins. Rb also strongly induced Integrin α10 (Itga10) in MC3T3 osteoblasts, and assays using Itga10 promoter/luciferase constructs showed that this induction is transcriptional and independent of Rb's phosphorylation status. Point mutagenesis and progressive deletion of the Itga10 promoter allowed us to identify an Rb-responsive region from -108 to -55 bp relative to the transcriptional start site. We also characterized a second mechanism linking Rb with cell adhesion. This mechanism requires the Rb-mediated repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when upregulated during carcinogenesis. Rb transcriptionally represses Pak1, as demonstrated by our nuclear run-on assays. Our Chip analyses showed an Rb-E2F complex binding to an Rb-responsive site in the Pak1 promoter that is rich in E2F binding sites, showing that Pak1 is an E2F target and suggesting that Rb's repressive effect on Pak1 consists of blocking E2F's transactivating activity on Pak1. Taken together, our results show that Rb promotes cell adhesion by inducing the expression of the cadherins and integrins necessary for cell adhesion to other cells and to a substrate, and by antagonizing the E2F-induced expression of cell adhesion destabilizers such as Pak1. Based on our data, we propose that Rb loss in OS exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis.
Citation Format: Brienne Engel, Pedro G. Santiago-Cardona, Bernadete Sosa-García, Douglas Cress. Molecular mechanisms of aggressiveness of osteosarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2013-206