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Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

A Corrigendum to this article was published on 01 April 2018

This article has been updated

Abstract

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

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Figure 1: Identification of Met-VELs and Met-VEL clusters through H3K4me1 ChIP–seq.
Figure 2: Met-VELs modulate gene expression during metastatic colonization of the lung.
Figure 3: Results from an in vivo high-throughput RNAi functional assay of candidate metastasis dependency genes.
Figure 4: F3 mediates lung metastasis of osteosarcoma.
Figure 5: Deletion of a single gained Met-VEL blunts F3 expression and mitigates lung metastasis of osteosarcoma cells.

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  • 07 February 2018

    In the version of this article initially published, two of the authors are incorrectly identified as John Stamatoyannopolus and Henri Versteeg. The authors' names are John A Stamatoyannopoulos and Henri H Versteeg. Also, the affiliation "Research Laboratory, Istituto Ortopedico Rizzoli, Bologna, Italy" is incorrect. The correct affiliation is "Laboratory of Experimental Oncology, Istituto Ortopedico Rizzoli, Bologna, Italy". The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors thank the members of the Tesar, Kaplan, and Helman laboratories for their input throughout the course of the project as well as B. Decker for his input on the manuscript text. Additional support was provided by the Genomics Core Facility of the Case Western Reserve University School of Medicine's Genetics and Genome Sciences Department and the Case Comprehensive Cancer Center (P30CA043703). This work was supported by the Liddy Shriver Sarcoma Initiative (P.C.S., C.K., J.J.M.), the QuadW Foundation (P.C.S.), Sarcoma Foundation of America (P.C.S.), St. Baldrick's Foundation (A.Y.H.), Alex's Lemonade Stand Foundation (A.Y.H.), Hyundai Hope-on-Wheels Program (A.Y.H.), Pediatric Cancer Research Foundation (A.Y.H.), CCCC AYA Oncology Pilot Grant (A.Y.H.), National Institutes of Health (NIH) grants F30 CA186633 (J.J.M.), F30 CA183510 (T.E.M.), T32 GM007250 (J.J.M., T.E.M., S.H.), R01CA193677 (P.C.S.), R01CA204279 (P.C.S.), R01CA160356 (P.C.S.), F31CA192874 (F.A.), R21CA218790 (A.Y.H.), NIH Intramural Visiting Fellow Program 15335 (M.M.L.), and NIH Intramural Research Program (C.K.).

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Contributions

J.J.M., C.K., and P.C.S. conceived the overall experimental design. J.J.M., C.F.B., and G.D. generated ChIP–seq, RNA-seq, and DHS-seq data. J.J.M., A.S., S.H., and P.C.S. completed analyses of ChIP–seq, RNA-seq, and DHS-seq data. J.J.M. and T.E.M. designed and completed the shRNA screening experiment and subsequent analysis. T.E.M. completed functional enrichment analysis of RNA-seq data. J.J.M. and I.B. generated 4C-seq data. J.J.M. and A.S. analyzed 4C-seq data. J.J.M., A.M., and I.B. and M.M.L. completed the in vivo and ex vivo metastasis experiments. J.J.M., J.T.M., and F.A. designed and completed the orthotopic metastasis experiments. J.J.M., D.R.C., and A.P.W.F. designed and completed the TALEN deletion experiments. M.Y.K. completed the in vitro F3 experiments. M.G., A.R., and P.P. provided subject tumor samples and clinical data. B.P.R. assessed F3 staining in subject tissue microarrays. A.D., A.Y.H., P.S.M., L.J.H., H.H.V., J.A.S., C.K., and P.C.S. provided the technical expertise and facilities to complete the experiments. J.J.M. and P.C.S. analyzed all data and wrote the paper. All authors provided intellectual input, edited, and approved the final manuscript.

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Correspondence to Peter C Scacheri.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures & Tables

Supplementary Figures 1–13 and Supplementary Table 1 (PDF 2043 kb)

Life Sciences Reporting Summary (PDF 162 kb)

Supplementary Table 2

Met-VEL gene overlaps across patient tumors and cell lines (XLSX 468 kb)

Supplementary Table 3

Hairpins used in high-throughput in vivo RNAi screen (XLSX 37 kb)

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Morrow, J., Bayles, I., Funnell, A. et al. Positively selected enhancer elements endow osteosarcoma cells with metastatic competence. Nat Med 24, 176–185 (2018). https://doi.org/10.1038/nm.4475

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