Abstract A33: Metastasis score expression profiling of isogenic cell lines with defined oncogenic mutations and upon treatment with an inhibitor of PI3K/PTEN signaling pathway

Abstract

Abstract Background: Human derived cell lines represent an important model system for fundamental research and translational medicine. We sought to gather patient-relevant information by examining a previously published metastasis score (MS) (proliferation index) for operable breast cancer in well-characterized isogenic cell lines. We chose to examine the impact on MS of specific genetic alterations in known oncogenes and consequence of treatment with a class I PI3K inhibitor. Investigation of the proliferation index of cells without the complexity of cell mixtures found in tumor microenvironment may shed light on tumor cell-specific transcriptional dysregulation. Methods: Nontumorigenic MCF10A and isogenic cell lines harboring (G12V), PI3Kα (H1047R) and p53 (null) mutations were cultured in T75cm tissue culture flasks overnight. MCF10A and isogenic PI3Kα (H1047R) cell lines were seeded and allowed to adhere overnight before addition of 0.3 µM, 1 µM and 3 µM GDC-0941 or DMSO control and incubated for 24 hours. Total RNA was prepared using RNeasy Kit (Qiagen) from cells. Expression profiling was carried out using RT-PCR for the previously described metastasis score (Tutt et al, 2009) that includes 14 genes and 3 reference genes. Results: MCF10A and the isogenic mutant cell lines had equivalent levels of proliferation as measured by MS and similar phenotypes under these monolayer culture conditions with high levels of growth factors. Modest dose-dependent decreases in the expression level of the constituent MS genes (1.5-3 fold) were observed with GDC-0191 exposure that translated into a marked decrease in overall MS (14 to 4) due to their equally weighted additive impact. The four genes that demonstrated the greatest decrease were CCNB1, BUB1, MYBL2, and UBE2S. These genes are highly associated with cyclin-dependent kinase (CDK1), a key player in cell cycle regulation. The MS score for these isogenic lines are also being defined under in vitro assay conditions that more accurately mimic the tumor microenvironment; in particular a 3-dimentional Matrigel assay that unleashes a marked differential metastatic/invasive phenotype in mutant vs. WT cells. Conclusion: MCF10A and isogenic cell lines harboring KRAS (G12V), PI3Kα (H1047R) and p53 (null) mutations had equivalent levels of proliferation as measured by MS. These results suggest that a combination rather than a single oncogenic mutant are required to alter the MS, although in vitro conditions likely play an important role in fully unmasking genotype-specific phenotypes in these clean gain-of-function models. Exposure of parental and PI3K (H1047R) cell lines to PI3K inhibitor GDC-0941 led to a dose-dependent decrease in constituent genes and composite MS. These results suggest that even though genes in the PI3K/PTEN pathway are not included in the molecular score, perturbation of this signaling pathway leads to alteration of the MS, by affecting cell cycle progression. Future studies are required to determine if alteration of MS in cell lines treated with different targeted oncogenic pathway inhibitors will recapitulate patient tumor response. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A33.