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Chromosome Instability Drives Phenotypic Switching to Metastasis

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Chromosome Instability Drives Phenotypic Switching to Metastasis Chromosome instability drives phenotypic switching to metastasis ChongFeng Gaoa,1, Yanli Sua, Julie Koemanb, Elizabeth Haaka, Karl Dykemab, Curt Essenberga, Eric Hudsonb, David Petilloc, Sok Kean Khood, and George F. Vande Woudea,1 aLaboratory of Molecular Oncology, Van Andel Research Institute, Grand Rapids, MI 49503; bCore Technologies and Services, Van Andel Research Institute, Grand Rapids, MI 49503; cLaboratory of Interdisciplinary Renal Oncology, Van Andel Research Institute, Grand Rapids, MI 49503; and dDepartment of Cell and Molecular Biology, Grand Valley State University, Grand Rapids, MI 49503 Contributed by George F. Vande Woude, November 3, 2016 (sent for review October 5, 2016; reviewed by Webster K. Cavenee and Peter K. Vogt) Chromosome instability (CIN) is the most striking feature of human successively isolating mesenchymal variants from clonal epithelial cancers. However, how CIN drives tumor progression to metastasis populations, and then isolating epithelial revertants of the mesen- remains elusive. Here we studied the role of chromosome content chymal variants. We have shown that generation of mesenchymal changes in generating the phenotypic dynamics that are required variants associated with loss of chromosome contents harbors genes for metastasis. We isolated epithelial and mesenchymal clones from encoding IJ protein, whereas generation of epithelial variants was human carcinoma cell lines and showed that the epithelial clones frequently caused by Zeb1 (zinc-finger E-box–binding homeobox 1) were able to generate mesenchymal variants, which had the haploinsufficiency through 10p loss. potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then Result and Discussion epithelial phenotypes recapitulated the steps in tumor progression OVCAR5 (OV5-P) cells growing in a Petri dish exhibited a het- to metastasis. Importantly, the generation of mesenchymal variants erogeneous morphology (Fig. 1A, “2D”). Two major cell types from clonal epithelial populations was associated with subtle were recognized: epithelium-like cells that formed compact islets, changes in chromosome content, which altered the chromosome and mesenchymal-like cells that grew in a scattered pattern. When transcriptome and influenced the expression of genes encoding cultured in 3D Matrigel (Fig. 1A, “3D”), epithelium-like cells intercellular junction (IJ) proteins, whereas the loss of chromosome generated hollow acini, and mesenchymal-like cells formed grape- 10p, which harbors the ZEB1 gene, was frequently detected in ep- like aggregates. We isolated three epithelial (OV5-E1,OV5-E2, ithelial variants generated from mesenchymal clones. Knocking and OV5-E3) and three mesenchymal (OV5-M1,OV5-M2,and down these IJ genes in epithelial cells induced a mesenchymal phe- OV5-M3) clones on the basis of their phenotype on Matrigel. The notype, whereas knocking down the ZEB1 gene in mesenchymal clones maintained their original phenotype on cell passage (Fig. 1 cells induced an epithelial phenotype, demonstrating a causal role B and C). Immunofluorescent staining with anti-epithelial cad- of chromosome content changes in phenotypic determination. Thus, herin (E-cadherin) antibody indicated that epithelial clones had our studies suggest a paradigm of tumor metastasis: primary epi- intact adherens junctions, which was not the case for the mesen- thelial carcinoma cells that lose chromosomes harboring IJ genes chymal clones (Fig. 1D). The mesenchymal phenotype of mesen- acquire an invasive mesenchymal phenotype, and subsequent chro- chymal clones was further confirmed by high levels of ZEB-1 and mosome content changes such as loss of 10p in disseminated mes- vimentin protein (Fig. 1E). Mesenchymal clones were more in- enchymal cells generate epithelial variants, which can be selected vasive than epithelial clones, as shown by an in vitro invasion assay for to generate epithelial tumors during metastatic colonization. (Fig. 1F). Despite their distinct phenotypes, all epithelial and mesenchymal clones displayed an identical DNA fingerprint tumor metastasis | chromosome instability | aneuploidy | clonal evolution | epithelial–mesenchymal transition (E-MT) Significance hromosome instability (CIN), defined by an elevated rate of Chromosome instability and its resulting karyotypic heteroge- Cchromosome missegregation and breakage, results in diverse neity make up one of the most striking characteristics of hu- chromosome abnormalities in tumor cell populations (1–7). Ac- man cancers. Yet whether chromosome loss or gain drives cumulating cytogenetic analyses of more than 60,000 cases of tumor progression to metastasis remains unknown. Here we human cancer have indicated that most of the solid tumors contain show that clonal populations of epithelial cells spontaneously chromosome aberrations, with each tumor displaying a distinct generate mesenchymal variants. These variants have potential abnormal karyotype (Mitelman database: cgap.nci.nih.gov/ for reverting to an epithelial phenotype. Importantly, we show MEDICAL SCIENCES Chromosomes/Mitelman). In typical human cancers, one-quarter of that the successive phenotypic variants selectively eliminate or the genome was affected by arm-level copy-number aberrations (8). acquire chromosome segments that harbor genes encoding Moreover, cancer genome sequencing revealed dynamic chromosome intercellular junctional proteins and their regulators. Thus, tu- content changes during clonal evolution of the tumor cell population mor metastasis can be a clonal process driven by chromosome (9–12). However, how chromosome loss or gain drives tumor pro- instability. gression to metastasis remains elusive (13–17). Tumor metastasis is a multistep process, with the acquisition of an invasive mesenchymal Author contributions: C.G. and G.F.V.W. designed research; C.G., Y.S., J.K., E. Haak, C.E., E. Hudson, D.P., and S.K.K. performed research; C.G., K.D., and G.F.V.W. analyzed data; phenotype being a crucial step for tumor dissemination, as is the and C.G. and G.F.V.W. wrote the paper. reacquisition of an epithelial phenotype for metastatic colonization Reviewers: W.K.C., Ludwig Institute for Cancer Research University of California, San (18, 19). Although clonal evolution theory has been well established Diego; and P.K.V., The Scripps Research Institute. as a general mechanism of tumor progression (16, 20), how it con- The authors declare no conflict of interest. tributes to the sequential phenotype acquisition leading to metastasis Freely available online through the PNAS open access option. remains unknown. We reasoned that CIN might drive the phenotypic 1 To whom correspondence may be addressed. Email: [email protected] or variations by selectively eliminating or acquiring chromosome seg- [email protected]. ments that harbor genes encoding IJ proteins and their regulators. To This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. test this hypothesis, we have recapitulated the metastatic process by 1073/pnas.1618215113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1618215113 PNAS | December 20, 2016 | vol. 113 | no. 51 | 14793–14798 Downloaded by guest on September 27, 2021 1 23 A 2D 3D BC1 23 E Acini E Grape -like M M D IF: anti-E-cadherin E F 10 ) E E E -2 1 2 3 8 E1 E2 E3 M1 M2 M3 Zeb-1 6 E-Cad 4 M1 M2 M3 Vimentin 2 β-Actin Migrating cells (10 cells Migrating 0 PE1 E2 E3 M1 M2 M3 E1 E1M1 E1M2 E1M3 GIE1M1 H E1 E1M2 E2 Zeb-1 E1M3 E3 E-Cad OV5 M1 Vimentin M2 β-Actin E-Cad 2D M3 J E1 E2 E3 M1 M2 M3 E1M1 E1M2 E1M3 94% 100% 100% 92.5% 100% 93% 100% 11.5% 90.5% Fig. 1. Isolation and characterization of epithelial and mesenchymal clones from OVCAR5 cells. (A) Morphology of OVCAR5 cells cultured in a Petri dish (2D) or in 3D Matrigel (3D). (B) Isolated epithelial and mesenchymal clones maintained their morphology after cell passage in a Petri dish. (C) Morphology of epithelial and mesenchymal clones cultured in Matrigel. (D) Immunofluorescent staining with anti–E-cadherin antibody showing adherent junctions in ep- ithelial, but not in mesenchymal, clones. (E) Western blot assay showing that epithelial and mesenchymal markers partitioned between the epithelial and mesenchymal clones, respectively. (F) Mesenchymal clones were highly invasive, as assayed in Matrigel chambers. (G) Diagram showing the pedigree of clones derived from OV5-P. (H) Characterization of mesenchymal variants from epithelial clone OV5-E1.(Top) Morphology when cells were cultured in Petri dishes. (Bottom) Immunohistochemical staining showing the loss of E-cadherin in mesenchymal variants. (I) Western blot assay showing partition of epithelial and mesenchymal markers between OV5-E1 and its mesenchymal variants. (J) FISH analysis with a probe covering the CDH1 gene. The numbers under each panel indicate percentage of interphase cells that contained signals shown in the images. pattern, indicating their common origin (SI Appendix, Table S1). mesenchymal variants grew in a scattered pattern with the loss of Spectral karyotyping (SKY) analysis indicated that both epithelial E-cadherin in immunofluorescent staining (Fig. 1H), as well as a and mesenchymal clones shared several derivative chromosomes gain of ZEB1 in Western blot analysis (Fig. 1I). with their parental OV5-P cells, which further proved their com- To evaluate the role of CIN in generating these mesenchy- SI Appendix mon origin ( , Table S2 and Fig. S2). Although the mal variants, we used fluorescence
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