Abstract A02: pRb-loss causes widespread changes to the transcriptome and proteome of cells

Abstract

Abstract Loss of the Retinoblastoma 1, pRb, tumor-suppressor gene is widespread in tumor cells and causes de-regulation of the E2F transcription factors. Aberrant E2F activity results in the over expression of genes required for proliferation. To determine how pRb-loss changes cells, we depleted pRb from RPE cells using shRNAs and measured the effect on the transcriptome and proteome using RNA-sequencing and quantitative Mass Spectrometry. These approaches produced a number of unexpected findings including; (A) many E2F target genes are transcribed but poorly translated, (B) pRb-loss induces a strong interferon response, and (C) pRb-deficiency increases the protein levels of the extracellular matrix components. In addition, our results support previous reports linking pRb to mitochondrial activity and metabolic reprogramming. The changes to the transcriptome and proteome are extensive and complex and we are currently conducting an in-depth analysis of these results however our preliminary analysis suggests that loss of the pRb tumor-suppressor gene causes widespread and previously unappreciated changes in the transcriptome and proteome of cells. To begin to understand the mechanisms regulating these changes in translation potential, we have begun assessing the post-transcriptional mechanisms controlling the levels of the E2F-target genes. This group of genes is induced upon pRb-loss however no associated increase in protein levels is detected. To identify the mechanisms controlling the translation potential of this collection of genes, we search for enriched motifs of post-transcriptional regulators in these mRNAs. This analysis found a strong enrichment for the Pumilio (PUM) RNA-binding protein. RNA-immunoprecipitation experiments confirm that the levels of E2F target genes bound by PUM increase upon pRb-loss. To assay how PUM levels regulate the protein levels of the E2F target genes, we depleted PUM1 or PUM2 using siRNAs and conducted quantitative Mass Spectrometry. From this analysis, we find a strong increase in the levels of some E2F target genes upon PUM and pRb-loss. Collectively, these results are providing new insights into the proteomic changes induced after pRb-loss and is highlighting some of the post-transcriptional mechanisms buffering these effects. Citation Format: Wayne O. Miles. pRb-loss causes widespread changes to the transcriptome and proteome of cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A02.