A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes

Abstract

The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.

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Figure 1: A Sleeping Beauty–dependent inactivatable Pten allele for coupled whole-body mutagenesis and tumor suppressor discovery in mice.
Figure 2: Characterization of Pten-inactivated prostate cancer and identification of genes that potentially drive its progression.
Figure 3: Perturbed biological pathways, cellular processes and previously uncharacterized tumor suppressor candidates enriched among the PSB∩PISB CIS genes.
Figure 4: Genetic inhibition of tumor suppressor genes drives prostate cancer progression through canonical signaling pathways.
Figure 5: Clinical significance of validated genes in human prostate cancer.
Figure 6: In vivo validation of Wac as a new obligate haploinsufficient gene in prostate cancer.

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Acknowledgements

We thank the staff members of the Research Support Facility at the Wellcome Trust Sanger Institute, the Laboratory of Molecular Medicine at IMOMA, and those at the Transgenic Animal Unit, the Molecular Histopathology Unit, the Department of Biochemistry and Molecular Biology and the Biobank of the Principality of Asturias at IUOPA, for excellent technical assistance. This work was supported by grants from the Wellcome Trust (grant no. 098051; A.B.), the Ministerio de Economía y Competitividad–Spain (grant no. SAF2014-52413; C.L.-O.) and the German Research Society (grant no. SFB1243; R.R.), as well as by funding from the Fundación María Cristina Masaveu Peterson (J.C.), the Fundación Centro Médico de Asturias (J.C.), the Fundación Bancaria Caja de Ahorros de Asturias/Liberbank (J.C. and A.A.), FEBS (J.d.L.R. and J.C.), CIBERONC (Plan Feder) (C.L.-O.), the Progeria Research Foundation (C.L.-O.), the EDP Foundation (C.L.-O.) and the German Cancer Consortium (R.R.). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA). J.d.l.R. is a recipient of a FEBS Long-Term Fellowship and was a recipient of a fellowship from the Fundación María Cristina Masaveu Peterson during part of this work. J.C. was a recipient of a FEBS Long-Term Fellowship in the initial phases of this work.

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J.d.l.R., R.R., C.L.-O., A.B. and J.C. designed the study; J.d.l.R., J.W., R.R. and J.C. generated mouse alleles and cohorts, and performed experiments; J.d.l.R., J.W., L.R., Q.L., M.A.L., G.S.V., R.R. and J.C. performed mouse necropsies; A.A., M.S.F.-G., M.T.F.-G. and G.J.H. performed histopathological analysis; J.d.l.R., M.J.F., Y.L and H.P. did bioinformatics analyses; J.d.l.R., C.L.-O. and J.C. interpreted results; M.J.F., S.B.d.Q., I.N., E.M., A.S. and R.F. contributed to some of the experiments; R.R., C.L.-O., A.B. and J.C supervised the study; and J.d.l.R. and J.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Juan Cadiñanos.

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The authors declare no competing financial interests.

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de la Rosa, J., Weber, J., Friedrich, M. et al. A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes. Nat Genet 49, 730–741 (2017). https://doi.org/10.1038/ng.3817

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