Project description:We performed RNA-seq to examine RNA expression profiles during MCF10A-ER-Src cell transformation and upon knockdowns of transcription factors
Project description:siSTAT3 knockdown of a tamoxifen initiated, transformation inducible, breast cancer model system (MCF10A-ER-Src), with associated controls of EtOH and siNEG treatments. A description of MCF10A-ER-Src cells can be found here: www.encodeproject.org
Project description:We applied ribosome profiling and RNA sequencing to examine gene expression regulation during oncogenic cell transformation. One model involves normal mammary epithelial cells (MCF10A) containing ER-Src. Treatment of such cells with tamoxifen rapidly induces Src, thereby making it possible to kinetically follow the transition between normal and transformed cells. The other model consists of three isogenic cell lines derived from primary fibroblasts in a serial manner (Hahn et al., 1999). EH cell is immortalized by overexpression of telomerase (hTERT), and exhibits normal fibroblast morphology. EL cell expresses hTERT along with both large and small T antigens of Simian virus 40, and it displays an altered morphology but is not transformed. ELR cell expresses hTERT, T antigens, and an oncogenic derivative of Ras (H-RasV12).
Project description:Purpose: The goal of this study is to examine gene expression regulation at the transcriptional and translational levels in response to various forms of nutrient deprivation, and whether there are differences between isogenic transformed and non-transformed cells. Mehods: We apply high-throughput sequencing of ribosome footprints (ribosome profiling) and poly(A) RNA (RNA sequencing) in an isogenic pair of transformed (tamoxifen-treated) and non-transformed (ethanol control) MCF10A-ER-Src cells subjected to the metabolic stresses that differentially affect global protein synthesis (Figure 1): deprivation of glutamine (for 30min and 4 hours), glucose (4hours), cysteine/cystine (4hours) or leucine/isoleucine/valine (brach-chain aminoacids - BCAA) (4hours). The same experiments were also performed in transformed ER-Src MCF10A treated with torin1 (500nM) for 4 hours. Results: Genome-wide translational profiling of glutamine deprived ER-Src MCF10A cells (for 30 minutes) shows increased translation of uORFs-containing mRNAs and down-regulation of ribosomal protein mRNAs, which is followed by increased translation and transcription of cytokine and inflammatory mRNAs (after 4 hours of glutamine deprivation). The transcription and translation of inflammatory and cytokine mRNAs is also stimulated in response to 4 hours deprivation of glucose, cysteine/cystine and BCAA, with the extent of stimulation correlating with the i) decrease in global protein synthesis and ii) down-regulation of all translationally-repressed mRNAs or ribosomal protein mRNAs. Conclusions: Pro-inflammatory gene expression is associated with translational repression in response to short-term nutrient deprivation.
Project description:Nontransformed cells form heterotypic cadherin junctions with adjacent transformed cells to inhibit tumor cell growth and motility. Transformed cells must override this form of growth control, called contact normalization, to invade and metastasize during cancer progression. Heterocellular cadherin junctions between transformed and nontransformed cells are needed for this process. However, specific mechanisms downstream of cadherin signaling have not been clearly elucidated. Here, we utilized a b-catenin reporter construct to determine if contact normalization affects Wnt signaling in transformed cells. b-catenin driven GFP expression in Src transformed mouse embryonic cells was decreased when cultured with cadherin competent nontransformed cells compared to transformed cells cultured with themselves, but not when cultured with cadherin deficient nontransformed cells. We also utilized a layered culture system to investigate the effects of oncogenic transformation and contact normalization on gene expression and oncogenic Src kinase mediated phosphorylation events. RNA-Seq analysis found that the cadherin dependent contact normalization inhibited the expression of 22 transcripts that were induced by Src transformation, and increased the expression of 78 transcripts that were suppressed by Src transformation. Phosphoproteomic analysis of cells expressing a temperature sensitive Src kinase construct found that contact normalization decreased phosphorylation of 10 proteins on tyrosine residues that were phosphorylated within 1 hour of Src kinase activation in transformed cells. Taken together, these results indicate that cadherin dependent contact normalization inhibits Wnt signaling to regulate oncogenic kinase activity and gene expression, particularly PDPN expression, in transformed cells in order to control tumor progression.