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 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 for metastasis. We isolated epithelial and mesenchymal clones from encoding IJ , 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) , whereas the loss of chromosome generated hollow acini, and mesenchymal-like cells formed grape- 10p, which harbors the ZEB1 , 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-) 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 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- Migrating cells (10 cells Migrating 0

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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 in situ hybridization (FISH) karyotypes among the three epithelial clones or among the mes- analysis with a probe covering the E-cadherin gene (CDH1). All enchymal clones were similar, numerous chromosomal changes the epithelial clones (OV5-E ,OV5-E, and OV5-E )contained were observed between the two different types. 1 2 3 three copies of 16q, whereas two of the three M clones (OV5-M1 Although the epithelial clones exhibited a highly homogeneous J SI phenotype, rare mesenchymal-like cells were observed during and OV5-M2) contained two copies of that arm (Fig. 1 and Appendix clonal expansion. We isolated three of these phenotypic variants ,TableS3). Importantly, two of the three mesenchymal variants (E M and E M ) that derived from OV5-E showed a from the OV5-E1 clone (Fig. 1G) on the basis of their grape-like 1 1 1 3 1 morphology in 3D culture. The variants were termed OV5-E1M1, decreased copy number of CDH1, suggesting a role of 16q loss OV5-E1M2, and OV5-E1M3,with“OV5-E1” referring their origin in the down-regulation of E-cadherin and the generation of and “M” to their acquired mesenchymal phenotypes. All the mesenchymal variants.

14794 | www.pnas.org/cgi/doi/10.1073/pnas.1618215113 Gao et al. Downloaded by guest on September 27, 2021 Although the mesenchymal clones exhibited nearly homoge- dramatic increase of E-cadherin protein in both OV5-E1M1 and neous morphology when cultured in Petri dishes, small epithe- OV5-M2 cells, indicating that down-regulation of ZEB1 was lial-cell-like clusters were occasionally noticed. We reasoned that sufficient to drive the epithelial phenotype (Fig. 2 D and E). the single-cell-derived mesenchymal cell population might have Therefore, we tested whether the generation of epithelial phe- generated epithelial variants during cell division. Taking advan- notype was driven by loss of 10p. FISH analysis showed that 92.5% tage of the differential sensitivity to trypsinization between epi- of OV5-E1M1E cells and 75% of OV5-M2E cells contained one thelial and mesenchymal cells, we enriched the epithelial variants copy of 10p, which is one copy less than their parental cells (Fig. by continuingly culturing cells to confluence and then removing 2F and SI Appendix,TableS4). SKY assay revealed that OV5- the mesenchymal cells with controlled trypsinization. Thus, we E1M1 cells contained two copies of full chromosome10, whereas obtained two cell populations: OV5-E1M1E from OV5-E1M1, OV5-E1M1E cells had one copy of full chr10 and one copy of a G and OV5-M2E from OV5-M2 (Fig. 2A). Epithelial cells were derivative chromosome that contained only 10q (Fig. 2 ). In half dominant in both populations, as shown by the dramatic down- of the cases, the derivative chromosome was i(10)(q10), which regulation of ZEB1 and up-regulation of E-cadherin (Fig. 2 B resulted from head-to-head fusion of two 10q’s. In the other half, a and C). FISH analysis with probes covering CDH1 gene on 16q copy of 10q was joined to various other chromosome segments indicated that 100% of OV5-E1M1E and OV5-M2E cells con- including 22q, 20q, 3p/q(pter-q21), 19p, der(2;15)(p10;q10), and tained two copies of 16q, which was the same as their parental 14q (Fig. 2G). OV5-M2E also lost one copy of 10p, but 10q in- cells. This result excluded the possibility that the up-regulation of variably translocated to 7q in all 20 of the cells analyzed. The E-cadherin in the epithelial revertants was through regain of 16q fact that both revertants lost 10p, but reserved 10q in various (SI Appendix, Table S3). different ways, suggested that genes on 10p were pivotal for The ZEB1 gene, located at 10p11.2, codes for a protein that is a maintaining a mesenchymal phenotype, whereas the genes on transcriptional repressor of E-cadherin (21, 22). Knocking-down 10q might be essential for an epithelial phenotype or cell survival. of ZEB1 with siRNA induced an epithelial morphology with a Therefore, chromosome breakage and rearrangement makes it

A B C D OV5-E1M1 OV5-E1M1E 30 OV5-E1M1 OV5-E1M1E OV5-E1M1-C OV5-E1M1-Zeb-si1

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Zeb1 E-Cad γ-Tub

EG OV5-E1M1 OV5-M2 1 2 M 1 M Zeb1 OV5- E-Cad OV5-E γ-Tub E E 1 2 M OV5-E M OV5-E M E M

F 1 1 1 1 1 OV5- OV5-E MEDICAL SCIENCES

OV5-E1M1 OV5-E1M1E OV5-M2 OV5-M2E

10q 20q 7q 10p 22q 10p 10p 10p(p11.23-pter) OV5-M OV5-M E 10q 2 2 1q (p11-q31) 10q 10q 10q 10q 10q 10q ? (20) (10) (3) (3) (20) (20) 2p 3p 15q 10p15.1 14q 10p15.1 7q 3q21 19p 10q 10q 1q 10q 10q 10q 10q25.3 10q25.3 (1) (1) (1) (1) ?

Fig. 2. Loss of 10p in generating epithelial revertants from clonal mesenchymal cells. (A) Morphology of epithelial revertants cultured in a Petri dish. (B)Real-timeRT- PCR and/or Western blot assay showing loss of ZEB1 and gain of E-cadherin in epithelial revertants. (C) Membranous E-cadherin staining in epithelial revertants. (D)

Epithelial morphology in OV5-E1M1 and OV5-M2 cells treated with siRNA to ZEB1 (Zeb1-si1). (E) Western blotting showing E-cadherin induction after ZEB1 knock-down with siRNA in OV5-E1M1 and OV5-M2 cells. Cells were treated with control siRNA (Control) or two siRNA against ZEB1 (Zeb1-si1 and Zeb1-si2). (F) FISH analysis for copy numberoftheZEB1geneon10p11.2.(G) SKY assay on epithelial revertants and their parental cells; representative karyotypes are shown. Variations of chr10 from 20 metaphases are depicted in red frames below the karyotypes. Numbers in parentheses indicate the incidences of the above chr10 pattern in 20 karyotypes. Metaphase

FISH was performed to confirm head-to-head fusion of two 10q’sinOV5-E1M1E, and the identities of der containing chr10 in OV5-M2Ecells(Bottom red frame). FISH probe on 10q25.3 was labeled with red color. Probe on 10p11.2 for OV5-E1M1E and 10p15.1 for OV5-M2E were labeled with green.

Gao et al. PNAS | December 20, 2016 | vol. 113 | no. 51 | 14795 Downloaded by guest on September 27, 2021 possible to eliminate a smaller chromosomal region con- morphology in 3D culture in the presence of HGF/SF. From the taining target gene or genes through chromosome loss. Im- DU-E population, we further isolated three branching clones (DU- portantly, all the OV5-E1M1E cells retained a copy of der(21)t EMn) that maintained a branching phenotype after multiple cell pas- (1;21;11;13)(p11;p11.1q22;q13q25;q11), which was unique to sages (Fig. 3A). DU-E displayed a high level of E-cadherin as well as OV5-E1M1. Similarly, OV5-M2E and OV5-M2 shared unique i(8) other IJ proteins, which are markers for an epithelial phenotype (Fig. 3 (q10) and der(16)t(13;16)(q22;q11.2) derivative chromosomes and B and C and SI Appendix,Fig.S2). These epithelial markers were presented almost identical karyotypes except for chromosomal decreased dramatically in DU-M and DU-EMn cells. Both DU-M and changes that involved chr10 and chr7 (Fig. 2G). These observa- DU-EMn clones were more invasive than DU-E cells (Fig. 3D). tions confirmed that OV5-E1M1E and OV5-M2E were the prog- A FISH assay indicated that the copy number of 16q22 did not SI Appendix eny of OV5-E1M1 and OV5-M2, respectively. Together, these decrease in DU-M or DU-EM cells ( ,TableS5). To results demonstrated that OV5-E1M1 and OV5-M2 cells gener- determine whether changes in chromosomes harboring other IJ ated epithelial revertants by selectively eliminating 10p. genes could be involved in phenotype determination, we per- To seek another paradigm for CIN determination of the metastatic formed comparative genomic hybridization on DU-M and DU- phenotype, we studied DU145, a prostate carcinoma cell line that has EMn cells, using DU-E cells as a reference (Fig. 3E). Significant a heterogeneous phenotype whenculturedinPetridishes(SI Ap- decreases of 1q, 4p, 6p, 14q, and 18p/q were detected in DU-EMn pendix,Fig.S2). Parental DU145 cells formed hollow acinar structures cells. Comparative genomics microarray analysis indicated that the or loose cell aggregates when cultured in 3D Matrigel. In the presence chromosome content changes closely correlated with chromosome of hepatocyte /scatterfactor(HGF/SF),theloosecell transcriptome changes (Fig. 3E), which was consistent with our aggregates formed branching structures, which are more easily dis- previous report (23). These transcriptome changes were barely de- tinguishable from acini. We therefore isolated an acini-forming clone tected in DU-EE clones, suggesting that the changes were relevant (DU-E) and a branch-forming clone (DU-M) on the basis of their to a mesenchymal phenotype. A SKY assay indicated that decreases

A B CD

EF

GH

Fig. 3. Phenotypic characterizations of clones from DU145 cells (A) Branching morphogenesis assay. Cells cultured in 3D were stained with calcerin-AM and observed by confocal microscopy. (B) Western blotting showing the down-regulation of intercellular junction proteins in cells with a mesenchymal phenotype. (C) Immunofluorescence staining showing the loss of adherent junctions and tight junctions in mesenchymal clones; the counterstain was Hoechst 33342.(D) Invasion results in Matrigel chamber assay. (E) Comparative genomic hybridization and comparative genomics microarray analysis showing chromosome dosage and chromosome transcriptome changes (log2 transformed chromosome transcriptome ratio between indicated clones and DU-E); blue, decrease; red,

increase. (F) Chromosome content changes in DU-E, DU-M, and DU-EM1–3 cells. The chromosomes or der chromosomes that contain the indicated chromo- some content are listed. Loss of 1q, 4p, 6p, 14q, and 18p/q plus gain of 3q in mesenchymal clones resulted from complicated chromosome changes. (G) Knocking down of intercellular junction proteins in DU-E cells induced mesenchymal phenotype. Cells were treated with a control siRNA (NC), or a combi- nation of siRNAs to CGN, DSP, DSC2, and DSG2 (IJ). Down-regulation of E-cadherin protein was detected with Western blotting. (H) Immunofluorescence staining showing the loss of E-cadherin and cytoplasmic translocation of ZO-1 at 48 h after transfection with siRNA to CGN, DSP, DSC2, and DSG2 (IJ).

14796 | www.pnas.org/cgi/doi/10.1073/pnas.1618215113 Gao et al. Downloaded by guest on September 27, 2021 of 1q, 4p, 6p, 14q, and 18p/q resulted from the loss of der(1)t clones (Fig. 3B and SI Appendix, Fig. S3 and Table S10). Four of (1;4)(p11;p11);der(6;16)(q11;p11.2) and der(18)t(14;18)(q13;q12), these genes were on chromosomes having copy number decreases which was observed in DU-E (but not in DU-EMn)cells(Fig.3F (SI Appendix, Table S12). Although the copy number of the CDH1 and SI Appendix,TablesS6–S9 for detailed calculation of chro- gene did not decrease, vigorous rearrangement occurred between mosome content changes). Nonetheless, DU-E and DU-EMn cells 16q and various other chromosomes (SI Appendix,Fig.S4). To shared der(13)t(1;8;13;1;19), der(8)t(7;8)(p11.1;p11.1), and del(15), confirm that the chromosome-loss-associated down-regulation of which were not found in the DU-M and parental DU145 cells, IJ proteins contributed to the generation of DU-EMn clones from indicating the lineage relation between DU-E and DU-EMn cells DU-E, we knocked down the expression of CGN, DSP, DSC2, (SI Appendix,TableS8). and DSG2 in DU-E, using a combination of siRNAs to each gene Chromosome copy number changes dramatically influenced the (Fig. 3G). Cells treated with siRNA exhibited disrupted tight expression of their resident genes: 18 of the 19 significantly altered junctions and adherens junctions, as indicated by the cytoplasmic genes changed expression in accord with the change in their host location of ZO1 and the decrease of E-cadherin protein (Fig. 3H). chromosome (SI Appendix,TablesS10andS11). Down-regulation These results suggest the loss of chromosomes harboring IJ genes of six IJ genes, including CDH1 (E-cadherin), CGN (), can be a mechanism for generating a mesenchymal phenotype. CLDN7 (claudin7), DSP (desmoplakin), DSC2 (desmocollin2), Unlike with OV5-P cells, the in vitro selection of epithelial and DSG2 (desmoglein2), were found in DU-M and DU-EMn variants from DU145 mesenchymal clones was not feasible, partly

DU-EM E-Cad E-Cad A 2 B 1 C

E-MT M-ET 1 DU-E DU-EM DU-EM E M 1 1 T1 EM Tu - 3D DU-EM DU-M DU- DU-EM DU DU-P 3D 3 T1 T1 T1 E E T1 1 1

M-ET ME - DU-M DU-MET1 DU

Tu DU-ME DU-EM DU-EM

D

(3) (2) (1) (1) (1) (1) (1) DU-M 21q 5p 5p Composition 5p 2q 5q 9q 18p of der 3p der(5)t(5;21) der(5)t(5;9) der(3;5)t(3;2;5) der(18)t(5;18) T1 5p 5p 5p 5q

DU-ME i(5)(p10) Chr.5 (9) (1)

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β-Act MEDICAL SCIENCES 10p11.2 (Zeb-1) G

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siRNA C Zeb-1 1 16q 19p Zeb-1 10q 10q

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Fig. 4. Chromosome changes in generating an epithelial phenotype through tumorigenesis (A) Strategies for isolating DU-M and DU-EM1–3 mesenchymal clones through 3D culture, and subsequent phenotypic switching of DU-M and DU-EM1 in tumor xenografting. 3D, clones isolated from 3D culture; Tu, sublines from tumor xenografts. (B) Loss of branching morphogenesis in epithelial revertants. (C) Immunofluorescent staining with anti–E-cadherin antibody

showing return of adherent junctions in revertants. (D) Gain of 5q in DU-MET1 relative to DU-M cells. (Left) Representative pseudocolor SKY images of DU-M and DU-MET1 cells showing their highly related karyotypes. (Right) Variations of chr5 in DU-M and DU-MET1. Numbers in parenthesis indicate incidence of each karyotype in 10 metaphases. (E) Gain of 5q drives epithelial phenotype by up-regulating key genes. DU-MET1 cells were transfected with siRNA to , Kibra, CXXC5, or a combination of the three (combo). Cells were also transfected with siRNA to E-cadherin as positive control. Loss of epithelial

phenotype upon siRNA transfection was monitored by E-cadherin expression, as revealed by Western blotting. (F) Loss of 10p in DU-EM1ET1 relative to DU- EM1; red, FISH signals on 10p11.2 that covers the ZEB1 gene. (G) Knocking down of ZEB1 converts DU-EM1 cells to an epithelial phenotype.

Gao et al. PNAS | December 20, 2016 | vol. 113 | no. 51 | 14797 Downloaded by guest on September 27, 2021 because of the similar sensitivity to trypsinization between epi- confirm that 10p loss in DU-EM1ET1 contributedtoanepithelial thelial and mesenchymal clones. The fact that metastatic tumors phenotype, we treated DU-EM1 cells with siRNA against ZEB1 and frequently have an epithelial phenotype suggests epithelial cells found a dramatic increase of E-cadherin protein (Fig. 4G). These may have a growth advantage. We tested whether the isolated results showed that, as in OV5 cells, 10p loss also played a causal DU-M and DU-EM1 cellsswitchedtoanepithelialphenotype role in generating the epithelial phenotype of DU-EM1ET1 cells. through tumorigenesis when inoculated s.c. into SCID mice. From Taken together, our results demonstrate that tumor cells un- tumor xenografts of these two lines, we established the cell lines dergo constant structural and numerical chromosomal changes A DU-MET1 and DU-EM1ET1 (Fig. 4 ). Both cells exhibited a that lead to karyotype diversity in a clonal cell population. In predominantly epithelial phenotype, as judged by their morphol- epithelial cells, the loss of chromosome segments harboring genes ogy in 3D culture (Fig. 4B) and their expression of E-cadherin encoding IJ proteins results in down-regulation of IJ proteins and (Fig. 4C). SKY analysis revealed nearly identical karyotypes be- the generation of mesenchymal variants that facilitate tumor dis- D SI Appendix tween DU-M and DU-MET1 (Fig. 4 and ,TablesS13 semination. Subsequently, loss of chromosome fragments carrying and S14). However, all DU-MET1 cells contained three copies transcriptional repressor of IJ proteins, such as ZEB1 in dissem- of 5q, whereas chr5 alterations in DU-M were highly diverse, inated mesenchymal cells, leads to generation of epithelial variants D showing seven different karyotypic patterns in 10 cells (Fig. 4 ), that can be selected for in appropriate environments to generate and eight of these 10 cells contained one or two copies of 5q. We metastatic colonization. Thus, the sequential phenotypic changes further performed FISH analysis on a mixture of eight cell lines SI Appendix during tumor metastasis can be a process of Darwinian selection of established from tumor xenografts of DU-M ( ,Fig.S5) phenotypic variants generated by chromosome instability. and found that 79.5% of cells contained three or more copies of 5q, confirming that selection of cells with more copies of 5q was a Materials and Methods common event during tumorigenesis of DU-M. Cell Lines and Reagents. OVCAR5 and DU145 cells were obtained from the Transcriptome analysis identified 14 genes on 5q that were up- National Cancer Institute. The authenticity of the cell lines was proved by regulated in DU-MET1 cells and no genes that were down-regu- karyotyping. Unless specifically indicated, cells were cultured in RPMI me- lated (SI Appendix, Table S15). The OCLN gene, which encodes dium supplemented with 5% (vol/vol) FBS (Invitrogen). Mouse anti–E-cadherin occludin, a main component of tight junctions, was the only IJ (HECD-1), rabbit anti-desmoplakin, mouse anti- (3G132), and gene. The gain in 5q was also associated with the up-regulation of mouse anti–β-actin (AC-15) antibodies were purchased from Abcam. Mouse kidney and brain protein (KIBRA) (5q34), an activator of SAV1 anti–γ-tubulin was from Sigma-Aldrich. Mouse anti-desmocollin2/3 and that is part of the SWH inhibitor complex of β-catenin (24, 25). rabbit anti–ZO-1 were from Invitrogen. Antibody against ZEB1 (H-102), as Also up-regulated was CXXC finger protein 5 (CXXC5) (5q31.2), well as horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit which inhibits β-catenin (26). Because the role of β-catenin in cell secondary antibodies, were from Santa Cruz Biotechnology. invasion is well established, up-regulation of KIBRA and CXXC5 might also contribute to the epithelial phenotype of DU-ME . 3D Cell Culture and Phenotype-Based Subcloning. Cells were collected by tryp- T1 sinization and suspended in culture medium at a concentration of 5,000 cells/mL We therefore examined the effects of siRNA knock-down of Fifty microliters of cell suspension was mixed with an equal volume of Matrigel OCLN, KIBRA, and CXXC5 on the phenotype of DU-MET1 (BD Bioscience) and placed into 96-well plates. After 30 min incubation at 37 °C, cells. Our results showed that knocking-down of OCLN triggered cells were grown on regular culture medium for 5–7 d to allow the formation of a significant down-regulation of E-cadherin, and that this was true acini or grape-like aggregates. For isolation of epithelial or mesenchymal clones for KIBRA and CXXC5, to a lesser extent (Fig. 4E). from OVCAR-5, cells suspended in culture medium at low density were mixed The DU-EM1 clone and its derivative DU-EM1ET1 showed a with equal volume of Matrigel and seeded into 6-cm Petri dishes. For isolation of major difference in chr10: DU-EM1 cells possessed two copies of acini or branching structures from DU145, cells were cultured in 3D in the chr10 per metaphase, whereas DU-EM1ET1 contained one full presence of HGF/SF (100 ng/mL). Acini and branching or grape-like aggregates chr10 and one copy of der(10;16)(p11.2; p11.2) (Fig. 4F and SI growing in 3D Matrigel were selected and isolated. Cells derived from individual Appendix,TableS16). Because der(10;16)(p11.2; p11.2) comprises acini or aggregates were diluted to 5 cells/mL and seeded in 96-well plates. Wells containing single cells were marked and expanded to obtain clones. 10q and 16q, DU-EM1ET1 lost one copy of 10p relative to DU-EM1. Expression array analysis revealed that two genes in the 10p region ACKNOWLEDGMENTS. We thank Kay Koo for administrative support and were significantly altered in DU-EM1ET1 relative to DU-EM1: David Nadziejka for technical editing of the manuscript. This work was ZEB1 and SFMBT2 were both down-regulated (SI Appendix,Table supported by the Jay and Betty Van Andel Foundation. The funders had no S17), correlating with the reduction in 10p chromosome dosage. To role in study design, data collection, or manuscript preparation.

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