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Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-? protein levels and activity.

ABSTRACT: Estrogen receptor-? (ER?) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ER?-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ER? protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ER? protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ER? target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ER? levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ER? protein levels and transcriptional activity.

SUBMITTER: Bolt MJ

PROVIDER: S-EPMC4871123 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

Bolt M J MJ Stossi F F Callison A M AM Mancini M G MG Dandekar R R Mancini M A MA

Oncogene 20140120 2

Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor ce ...[more]

PMID: 24441042

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Project description:Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor. The brain-infiltrative character of glioblastoma makes complete surgical removal of the tumor impossible and neither radiation nor current chemotherapy provide cure. Recent evidence shows that glioblastoma multiforme consists of heterogeneous cell populations which differ in tumor-forming potential. Enriched tumor-initiating capacity has been linked to poorly differentiated glioblastoma cells sharing features with neural stem cells. Thus, these cells are important targets for new therapeutic strategies. We aim to identify novel targets controlling maintenance and differentiation in glioblastoma-initiating cells through high throughput screening. To this end, we utilized libraries of small chemical compounds and small interference RNAs in combination with automated imaging and data analysis. Patient-derived glioblastoma cells were expanded and characterized using neural stem cell conditions. In culture, the cells showed low differentiation but expression of neural stem cell markers such as Nestin and Sox2. Upon intracranial injection into SCID mice these cells gave rise to tumors displaying the hallmarks of the human disease. Differentiation of glioblastoma-initiating cells (for example elicited through bone morphogenetic protein, BMP) was associated with strong morphological changes. Hence, cellular morphology, as well as markers specific for differentiation or death were used as screen readout. Lentiviral RNA interference-based screening yielded several gene knockdowns leading to ‘forced’ differentiation of glioblastoma-initiating cells. For example, knockdown of TRRAP (transformation/transcription domain associated protein) led to strongly increased differentiation and loss of proliferative and self-renewing capacity in these cells. TRRAP is an adapter protein implicated in oncogenic transformation through c-MYC transcription activation, also participating in chromatin remodeling and DNA repair. Glioblastoma-initiating cells with reduced TRRAP displayed increased apoptosis upon treatment with the genotoxic agent temozolomide. In vivo, Trapp knockdown cells were not able to give rise to glioblastoma upon transplantation into the brain of SCID mice. Together, these findings support a crucial role for TRRAP in maintenance and tumorigenicity of glioblastoma-initiating cells and might offer future therapeutic options. Two treatments compared to control: two different shRNA sequences for TRRAP were compared to a control shRNA sequence in their effects on global transcription in brain tumor initiating cells

Dataset Information

Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-? protein levels and activity.

Publications

Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

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