Abstract A12: A retinoblastoma protein phosphorylation code associated with cell adhesion and invasiveness
Authors: Perez-Morales Jaileene , Gonzalez-Flores Jonathan , Rosa Alexander De La , Estrada Elvin , Santiago-Cardona Pedro 
Published: May 2016

Abstract
Lung cancer remains the leading cause of cancer deaths among men and women worldwide. Its five-year survival rate (17.8%) is by far the lowest among the most common cancers such as colon (65.4%), breast (90.5%), and prostate (99.6%). Early detection and screening still remain challenging, and despite the many recent breakthroughs in lung cancer treatment, improvement in the long-term survival of lung cancer patients is still limited. The inactivation of the retinoblastoma tumor suppressor (Rb) is a major driving force behind tumorigenesis in most cancer types, including lung cancer. Rb is a phosphoprotein traditionally known as a cell cycle repressor. While Rb inactivation in small cell lung carcinomas, osteosarcomas and retinoblastomas occurs by mutational inactivation of the RB1 gene, most other human cancers show Rb inactivation by hyperphosphorylation. The effect of Rb hyperphosphorylation in cell cycle control has been characterized and shown to consist in the abrogation of Rb’s capacity to bind and repress the EF2 transcription factors responsible for triggering S-phase related gene expression. However, our data show that Rb phosphorylation can have novel consequences beyond cell cycle control, and that certain phosphorylations in Rb can impair the cell’s capacity to engage in cell-to-cell interactions. In order to search for phosphorylation patterns that may be associated with cell adhesion defects, we conducted a mass spectroscopy analysis on a panel of E-cadherin-expressing and E-cadherin-deficient cell lines. This analysis identified a four-residues phosphorylation signature (S612, S249, S807 and S821) that is associated to lack of E-cadherin expression. These four residues are strongly phosphorylated in the E-cadherin null cell line H520, relative to the E-cadherin expressing cell line H1666. Consistent with these data, H520 cells overexpress the Cyclin-dependent kinases Cdk2 and Cdk6, both of them known to have Rb as one of its targets. Treatment with CDK inhibitors (Roscovitine, PD00332991, and GW8510) resulted in inhibition of Cdk2, Cdk5, Cdk7, and Cdk9 together with reduced the phosphorylation of Rb S807 and S612, and with the reestablishment of E-cadherin expression. Moreover, shRNA-mediated Rb knockdown caused a marked induction of EMT in our lung cancer cell lines, indicated by decreased E-cadherin expression and elevated N-cadherin levels. Taken together, our data show a Rb phosphorylation signature associated with a non-traditional role for Rb as a regulator of cell adhesion. Given that Rb phosphorylation in specific residues can lead to loss of E-cadherin expression and concomitant loss of cell adhesion, both of them landmarks of the metastatic phenotype, we postulate that this phosphorylation signature may have prognostic value in predicting a proclivity for metastasis of lung cancer cells.
Citation Format: Jaileene Perez-Morales, Jonathan Gonzalez-Flores, Alexander De La Rosa, Elvin Estrada, Pedro Santiago-Cardona. A retinoblastoma protein phosphorylation code associated with cell adhesion and invasiveness. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A12.