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Chromosome Instability, Chromosome Transcriptome, and Clonal Evolution of Tumor Cell Populations

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Chromosome Instability, Chromosome Transcriptome, and Clonal Evolution of Tumor Cell Populations Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations ChongFeng Gao*, Kyle Furge†, Julie Koeman‡, Karl Dykema†, Yanli Su*, Mary Lou Cutler§, Adam Werts*, Pete Haak¶, and George F. Vande Woude*ʈ Laboratories of *Molecular Oncology, †Computational Biology, ‡Germline Modification, and ¶Microarray Technology, Van Andel Research Institute, 333 Bostwick Avenue, N.E., Grand Rapids, MI 49503; and §University Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 Edited by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, and approved March 29, 2007 (received for review January 23, 2007) Chromosome instability and aneuploidy are hallmarks of cancer, Phenotypic switching is fundamental for malignant progression but it is not clear how changes in the chromosomal content of a cell (6), and therefore it is important to understand the responsible contribute to the malignant phenotype. Previously we have shown mechanisms. that we can readily isolate highly proliferative tumor cells and their Glioblastomas characteristically show extensive regional cytoge- revertants from highly invasive tumor cell populations, indicating netic heterogeneity (10, 11), and this diversity may be responsible how phenotypic shifting can contribute to malignant progression. for tumor evolution and progression (10, 12). Here we show that Here we show that chromosome instability and changes in chro- distinct changes in karyotype from chromosome instability accom- mosome content occur with phenotypic switching. Further, we pany phenotypic switching. These changes, in turn, dictate changes show that changes in the copy number of each chromosome in the chromosome transcriptome that provide the expression of quantitatively impose a proportional change in the chromosome individual genes that are necessary for the conversion between the transcriptome ratio. This correlation also applies to subchromo- invasive and proliferative phenotypes. somal regions of derivative chromosomes. Importantly, we show that the changes in chromosome content and the transcriptome Results and Discussion favor the expression of a large number of genes appropriate for Karyotype Differences Accompany Switching of Glioblastoma Tumor the specific tumor phenotype. We conclude that chromosome Cell Phenotypes. To determine whether chromosome instability is instability generates the necessary chromosome diversity in the responsible for tumor cell phenotypic switching, we examined tumor cell populations and, therefore, the transcriptome diversity DB-P, DB-A2, DB-A6, and A2-BH7 cells (SI Table 4) by using to allow for environment-facilitated clonal expansion and clonal spectral karyotyping (SKY) (SI Fig. 4). For each cell type, we evolution of tumor cell populations. determined the total number of copies of each chromosome [or derivative (der) chromosomes] on the basis of 10 metaphase cells aneuploidy ͉ glioma ͉ invasion ͉ proliferation ͉ HGF/SF (Tables 1 and 2, respectively). Each cell population had near- tetraploid karyotypes, but karyotypes were particularly different from the parental DB-P cells as well as distinct from one another owell first proposed the clonal evolution of tumor cell popu- (Tables 1 and 2). Nlations to explain how malignant tumors arise over time (1). Tumor progression results from genetic variability within the tumor The Differences in DB-A2 and DB-P Cell Karyotypes Are Reflected in cell population that allows for clonal expansion of more aggressive Their Transcriptome Ratios. The significant differences in the karyo- tumor phenotypes (1, 2). Although chromosome instability and the types of each subclone led us to ask whether the changes in resulting cytogenetic heterogeneity are the most readily recognized chromosome content mediated changes in chromosome transcrip- genetic events associated with tumor progression (3–5) and may be tome that could influence phenotype determination. Recent gene responsible for tumor evolution and progression, precisely how they expression profiling studies have been used to assess the influence contribute to the malignant phenotype is not clear. Invasion and of chromosomal imbalance on overall gene expression (13–17). We proliferation are crucial requirements for tumor progression, and used this approach to determine whether the ratio of the changes we have chosen to study these steps in glioblastoma tumor cells (6). in chromosome content between parental DB-P cells and the Glioblastoma cells invade normal brain tissue (7); after surgical subclones influenced the chromosome transcriptome ratios. Begin- resection, residual invasive cells can quickly regain a proliferative ning with cDNA microarrays, individual gene expression differ- phenotype, progressing to a more aggressive tumor (8). Because ences between the DB-A2 subclone and the parental line, DB-P, glioblastomas rarely metastasize from the CNS, the sequential were calculated as described (15). The transcriptome ratios were selection of invasive and proliferative tumor cells constitutes the determined by averaging the relative expression ratios of each gene main theme for this tumor’s progression (7). It is, therefore, in a given chromosome or der subchromosomal region. These data MEDICAL SCIENCES critically important to understand the molecular mechanisms that are presented as log2-transformed to show the direction (ϩ/Ϫ) and permit high-frequency phenotypic switching and control tumor cell magnitude or are in linear scale to compare to the chromosome proliferation and invasion (6). content ratio. The log2-transformed data were displayed as scatter c-Met and its ligand, hepatocyte growth factor/scatter factor plots on the respective chromosome (Fig. 1). In this way, the (HGF/SF), can regulate both proliferative and invasive phenotypes of glioblastoma tumor cells (9). We have previously shown that alternating between proliferative and invasive phenotypes was Author contributions: C.G. and G.F.V.W. designed research; C.G., J.K., Y.S., A.W., and P.H. critically linked with switching between the Myc and Ras/MAPK performed research; C.G., K.F., K.D., and M.L.C. analyzed data; and C.G. wrote the paper. pathways, respectively (6). Starting with a highly invasive popula- The authors declare no conflict of interest. tion, DB-P, we were able to select two subclones, DB-A2 and This article is a PNAS Direct Submission. DB-A6, that are highly proliferative or both invasive and prolifer- Abbreviations: der, derivative; HGF/SF, hepatocyte growth factor/scatter factor; SKY, spec- ative, respectively. The parental DB-P cells and each subclone tral karyotyping; uPA, urokinase-type plasminogen activator. showed distinct in vitro and in vivo phenotypes and signaling ʈTo whom correspondence should be addressed. E-mail: [email protected]. pathways that correlated with their invasive or proliferative phe- This article contains supporting information online at www.pnas.org/cgi/content/full/ notypes [supporting information (SI) Table 4]. From the DB-A2 0700631104/DC1. subclone, we further selected a highly invasive revertant, A2-BH7. © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700631104 PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 ͉ 8995–9000 Downloaded by guest on September 30, 2021 Table 1. Full chromosomes in DB-P and its subclones via SKY subclone and the parental DB-P (Table 1 and Fig. 1A). Concor- Chromosome DB-P DB-A2 DB-A6 A2-BH7 dantly, a comparison of the chromosome transcriptomes shows that the average of the number of up- and down-regulated genes on 1 2(10) 20 2(10) 20 2(10) 20 2(10) 20 these chromosomes was largely unchanged (0.08, Ϫ0.02, 0.01, and 2 3(10) 30 4(5) 3(5) 35 3(9) 2(1) 29 4(7) 3(3) 37 0.02 in log2 ratio units, respectively) (Fig. 1A). However, when the 3 3(10) 30 4(1) 3(9) 31 3(10) 30 2(10) 20 chromosome copy number changed between DB-A2 and DB-P, the 4* 0 000 5 2(10) 20 2(9) 1(1) 19 2(7) 1(3) 17 2(10) 20 chromosome transcriptome also changed in the same direction. 6 3(10) 30 3(10) 30 3(6) 2(4) 26 2(10) 20 Thus, for chromosomes 2, 7, and 8, which were higher in copy 7 3(10) 30 4(10) 40 5(1)3(4) 2(5) 27 3(10) 30 number in DB-A2 cells, the transcriptome ratios were 0.18, 0.16, 8 3(10) 30 4(10) 40 4(7) 3(3) 37 4(10) 40 and 0.30 in log2 ratio units, respectively (Fig. 1B). For chromosomes 9 3(9) 2(1) 29 2(10) 20 3(1)2(7) 1(2) 19 2(10) 20 9, 14, 15, 20, and 21, which were fewer in copy number in DB-A2, 10 3(10) 30 2(10) 20 2(10) 20 2(10) 20 the corresponding ratios were Ϫ0.21, Ϫ0.33, Ϫ0.25, Ϫ0.32, and 11* 4(2) 3(6) 2(2) 30000Ϫ0.54, respectively (Fig. 1B); chromosome 21 showed the greatest 12 4(10) 40 4(9) 3(1) 39 4(8) 3(2) 38 4(10) 40 difference in copy number and the largest change in transcriptome 13 3(9) 2(1) 29 2(8) 1(2) 18 2(10) 20 2(10) 20 14 3(7) 2(3) 27 2(10) 20 2(10) 20 2(10) 20 ratio. These results show remarkable concordance between the 15 5(5) 4(3) 3(2) 43 4(8) 3(2) 38 3(9) 2(1) 29 4(10) 40 chromosome copy number and the change in chromosome tran- 16 1(10) 10000scriptome, suggesting that the change in chromosome content can 17 4(10) 40 4(9) 3(1) 39 4(7)3(2)2(1) 36 4(10) 40 be responsible for the changes in expression of each gene as part of 18 3(9) 2(1) 29 3(10) 30 3(10) 30 3(10) 30 the transcriptome. 19 4(3) 3(7) 33 4(10) 40 4(10) 40 4(10) 40 20 5(8) 4(2) 48 4(9) 3(1) 39 4(10) 40 4(10) 40 Subchromosomal Regions of Derivative Chromosomes Contribute to 21 8(1) 7(7) 6(2) 69 4(9) 3(1) 39 4(10) 40 4(4) 3(6) 34 the Chromosome Transcriptome (DB-A2 vs.
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