Abstract C12: Molecular mechanisms of aggressiveness of Rb-deficient tumors
Authors: González-Flores Jonathan , Cruz-Cosme Ruth , Rodríguez-Ortiz Yariana , Brady Sarah , Fraticelli-Rosado Ricardo , Engel Brienne , Cress Douglas , Santiago-Cardona Pedro G. 
Published: August 2014

Abstract
Osteosarcomas (OS) and small cell lung carcinomas (SCLC) are poorly differentiated, highly aggressive tumors, with an early proclivity to metastasize. Prognosis is poor for both, with only 10-20% of patients achieving long-term disease-free intervals. Still, OS and SCLC aggressivity remain to be molecularly explained. Interestingly, these cancer types show high rates of inactivation of the RB1 gene coding for the retinoblastoma protein (Rb), a known cell cycle repressor. This observation led us to hypothesize that in OS and SCLC, Rb loss during early tumorigenesis facilitates both increased proliferation and an early tendency for metastasis. Supporting our hypothesis, our previous studies in osteoblasts showed that Rb transcriptionally regulates a wide repertoire of cell adhesion genes, including those coding for adherens junction cadherins and integrins. We also found that adhrens junction assembly requires the Rb-dependent transcriptional repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when up-regulated. Thus, the early proclivity for metastasis of OS and SCLC may be related to perturbations in cell adhesion due to Rb loss.
Here we present new data using the SCLC line H187 and the non-small cell lung carcinoma (NSCLC) cell lines H1975 and H520. SCLC carcinomas have high rates (&gt; 90%) of Rb loss while NSCLC are known to be Rb wild type. Therefore, comparing H1975 and H520 vs. H187 provided us with an Rb-proficient vs. Rb-deficient system similar to the one we previously used to study Rb-engendered effects in osteoblasts. Our immunoblots and qRT-PCR showed that the Rb-null H187 cells expressed dramatically reduced levels of E-cadherin mRNA and protein relative to H1975 and H520, confirming that Rb loss impairs cell adhesion due to adherens junction loss. Interestingly, N-cadherin is up-regulated in H187 cells, suggesting that Rb loss triggers a cadherin switch similar to the one associated with epithelial-to-mesenchymal transitions. Other cell adhesion-related genes we found down-regulated in Rb-null H187 cells relative to H1975 and H520, as determined by qRT-PCR, were α-catenin, β-catenin, OB-cadherin, and the cytoskeletal adapter proteins eplin, vinculin, formin-1, and α-actinin. Our immunoblots also showed decreased levels of merlin, an adherens junction-interacting protein, in H187 cells. We also studied the expression of Rac1-binding proteins, given their involvement in cell adhesion. Our immunoblots and qRT-PCR showed reduced levels of IQGAP1 in H187 cells relative to H1975 and H520 cells. Given that IQGAP1 is a GTPase activating protein (GAP) that represses Rac1 by promoting its GDP-bound inactive state, we postulate that Rb represses Rac1 function via IQGAP1. Beta-2-chimaerin, another Rac1 GAP was also down regulated at the protein level in H187 cells. Taken together, our data in lung cell lines recapitulate most of our findings in osteoblasts, both in regards to the effect of Rb on cell adhesion and to the need for the Rb-mediated repression of Rac1 for the establishment of cell adhesion. Based on our data, we propose that Rb loss in OS and SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates aggressiveness of tumors at early stages by perturbing cellular adhesion, which in turn facilitates tumor cell detachment and metastasis.
Citation Format: Jonathan González-Flores, Ruth Cruz-Cosme, Yariana Rodríguez-Ortiz, Sarah Brady, Ricardo Fraticelli-Rosado, Brienne Engel, Douglas Cress, Pedro G. Santiago-Cardona. Molecular mechanisms of aggressiveness of Rb-deficient tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C12.