Abstract 2640: Inhibition of farnesylation induces senescence and apoptosis in osteosarcoma

Abstract

Abstract Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that osteosarcoma cell lines respond to the FTI tipifarnib with growth arrest, cellular enlargement, and flattened morphology, all changes consistent with the senescent phenotype (Hall et al., 2010). These observations led us to evaluate the ability of FTIs to induce senescence in osteosarcoma. Further, it was clear that cells retaining intact p53 responded at much lower concentrations of FTIs than did cells with absent/inactive p53, suggesting a possible role for p53 in this response. We measured endogenous β-galactosidase activity by in situ chromatography and quantified expression levels of common senescence markers including p53, p21Cip1, p16INK4a and DEC1 by densitometry analysis of western blot and qPCR in two human osteosarcoma cell lines, OS187 and Saos2. Cell growth was measured by direct counting of nuclei, and cell cycle was analyzed by PI staining and flow cytometry. Apoptosis was assessed by PARP cleavage, annexin V staining and flow cytometry. Variable responses to tipifarnib correlated with p53 status. In p53 proficient OS187 cells, 24h exposure to very low concentration of tipifarnib (10−9 M) induced upregulation of DEC1 and a 45-fold increase in p21Cip1 protein, accompanied by increased expression and phosphorylation of p53. This event also correlated with transient reduction of cyclin A, B1, and E proteins in a time-dependent manner, leading to cell cycle arrest which is comparable to tipifarnib-treated 293T cells. OS187 cells also exhibited elevated senescence-associated β-galactosidase activity in response to low-dose tipifarnib treatment after 24h. Meanwhile, Saos2 cells (p53-null) gave no response at 10−9 M tipifarnib. At higher concentrations (10−7 M), modest induction of p21Cip1 (10-fold increase) and p16INK4a (2-fold increase) proteins and mild inhibition of cell cycle progression were observed. Augmented senescence-associated β-galactosidase activity became detectable after Saos2 cells were exposed to high concentration of tipifarnib (10−6 M) for at least 48h. In addition to senescence, tipifarnib increased PARP cleavage and annexin V staining in OS187 cells, indicating that apoptotic cell death may also play a role in the anti-tumor activity of FTIs. These findings suggest that tipifarnib could efficiently promote senescence through a p53-dependent pathway at low concentrations, and via p53 independent pathways at higher concentrations. Induction of both cellular senescence and apoptosis may contribute to the growth inhibition induced by FTIs in osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2011-2640