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RNAi screens in mice identify physiological regulators of oncogenic growth

Abstract

Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here we tackle this problem by carrying out the first genome-wide RNA-interference-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover previously unknown as well as anticipated regulators of embryonic epidermal growth. Among the top oncogenic screen hits are Mllt6 and the Wnt effector β-catenin, which maintain HrasG12V-dependent hyperproliferation. We also expose β-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we validate functional significance in mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies.

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Figure 1: Embryonic epidermal tissue growth is rapid and responsive to oncogenic Hras.
Figure 2: Genome-wide RNAi screens for physiological regulators of normal and oncogenic growth identify expected and surprising regulators.
Figure 3: Suppressing β-catenin and Mllt6 selectively affects HrasG12V-dependent epidermal hyperplasia.
Figure 4: HrasG12V-induced epidermal growth affects other signalling pathways.
Figure 5: β-catenin and Mllt6 depletion impair HrasG12V-dependent tumorigenesis.

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Gene Expression Omnibus

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Raw RNAseq data can be accessed at Gene Expression Omnibus under accession number GSE48480 and permissions information is available at http://www.nature.com/reprints.

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Acknowledgements

We thank J. Fagin for inducible oncogenic Hras mice; S. Williams, M. Schober, A. Rodriguez Folgueras, S. Dewell and D. Schramek for intellectual input; D. Oristian and N. Stokes as mouse specialists; Comparative Bioscience Center (AAALAC accredited) for care of mice in accordance with National Institutes of Health (NIH) guidelines; Genomics Resource Center (C. Zhao, Director) for sequencing; Bioimaging Center (A. North, Director) for advice; Flow Cytometry facility (S. Mazel, Director) for FACS sorting. E.F. is an Investigator of the Howard Hughes Medical Institute. This research was supported by grants from the NIH (R37-AR27883 (E.F.) and K99-AR061469 (S.B.)), Emerald Foundation (E.F.) and Human Frontiers Science Program Postdoctoral Fellowship (S.B.).

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S.B., P.J. and E.F. designed the experiments. S.B. and P.J. made shRNA pools and lentivirus, and performed the screens. Illumina sequence analysis was done by E.H. and S.B. RNAseq and IPA analyses were performed by S.B., B.E.K. and P.J. Imaging was done by S.B. and N.O., and image analysis by S.B. and E.H. CHIP-seq data was generated by W.-H.L., and P.J. performed luciferase assays. S.B. and E.F. wrote the paper. All authors provided intellectual input, vetted and approved the final manuscript.

Corresponding author

Correspondence to Elaine Fuchs.

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The authors declare no competing financial interests.

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Beronja, S., Janki, P., Heller, E. et al. RNAi screens in mice identify physiological regulators of oncogenic growth. Nature 501, 185–190 (2013). https://doi.org/10.1038/nature12464

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