Abstract B13: R-spondin3 overexpression induces transformation and deregulates expression of genes controlling cell proliferation and apoptosis in NIH3T3 cells
Authors: Zimberlin Maria Noel , Rubinstein Natalia , Coso Omar A. , Kordon Edith C. 
Published: May 2012

Abstract
It has been reported that R-spondin3 (Rspo3) is a common insertion site for the viral genome in mouse mammary tumor virus (MMTV) induced-mammary tumors and that HC11 cells over-expressing this protein become tumorigenic when transplanted into mice. In addition, we have found that Rspo-3 over-expression is associated with loss of hormone regulation of initially pregnancy-dependent MMTV-induced mouse mammary tumors. In order to determine whether this gene may behave as an oncogen in culture, even in cells that do not derive from mammary tissue, NIH3T3 cells were stably transfected with mouse Rspo3 full-length cDNA under the control of the cytomegalovirus (CMV) promoter (3T3-Rspo3 cells). We intend this cell line to provide a tool that might allow the study of signaling pathways altered by Rspo-3 expression
We found that 3T3-Rspo3 cells showed the capacity to form foci when cultured on top of a lawn of normal NIH3T3 cells attached to plastic dishes and to grow as colonies when seeded in soft-agar. To determine signaling transduction pathways that might be altered by Rspo3 over-expression, we analyzed the level of expression of phosphorylated proteins critical in the regulation of cell growth or death and found that transfected cells showed higher levels of phosphorylated AKT and p38 MAPK, while no significant differences were found when the levels of other phosphorylated MAPKs (as Erk1/2) were analyzed. Besides, crystal violet staining assays revealed that Rspo3 transfected cells were less dependent on serum concentration to grow in culture. Interestingly, survival of 3T3-Rspo3 cells was reduced by treatment with a potent and selective phosphatidylinositol-3 kinase (PI-3K) pharmacological inhibitor, LY # 294002, a well-known regulator of signaling by AKT. In addition, expression of Cyclin D1, a protein associated with cell proliferation, was significantly increased and correlated with Rspo3 overexpression. To determine other genes that might be involved in 3T3-Rspo3 cell survival, we studied the level of expression of Bcl family members. We found that the cells over-expressing Rspo3 showed diminished expression of pro-apoptotic proteins BAD and BAX, comparing to control cells. On the other hand, we observed higher expression of the anti-apoptotic (long) Bcl-X form. Besides, preliminary results indicate that LY # 294002 treatments alter the expression pattern of Bcl family members at the protein level, suggesting that AKT phosphorylation might be involved in regulation of the balance between pro- and anti-apoptotic proteins.
In summary, we have developed a tool that can be used to dissect signaling pathways that may be responsible for the transforming effects triggered by over-expression of Rspo3. Our results, added to the reported in vivo studies, provide evidences that Rspo3 can be fully characterized as an oncogene. In addition, we have determined that activation of the PI-3K/AKT pathway might be involved in such an activity.
Citation Information: Cancer Res 2009;69(23 Suppl):B13.