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. 2018 Jul;32(7):3502-3517.
doi: 10.1096/fj.201700247RR. Epub 2018 Feb 8.

Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness

Darawalee Wangsa  1 , Isabel Quintanilla  2 , Keyvan Torabi  2   3 , Maria Vila-Casadesús  2   4 , Amaia Ercilla  5 , Gregory Klus  1 , Zeynep Yuce  1   6 , Claudia Galofré  2 , Miriam Cuatrecasas  7 , Juan José Lozano  4 , Neus Agell  5 , Daniela Cimini  8 , Antoni Castells  2 , Thomas Ried  1 , Jordi Camps  2   3
Affiliations

Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness

Darawalee Wangsa et al. FASEB J. 2018 Jul.

Abstract

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

Keywords: aneuploidy; colorectal cancer; genomic instability; invasive front; lagging chromosomes.

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Conflict of interest statement

The authors thank Z. Storchova (Technische Universität Kaiserslautern, Kaiserslauter, Germany) for kindly providing cell lines; A. Bosch and M. Calvo (University of Barcelona) for assessing the data analysis; and Z. Wong [U.S. National Institutes of Health (NIH)], R. Ebner (NIH), and S. D. Rutledge (Virginia Tech) for critical reading. The authors are also grateful to B. Chen (NIH) for editorial assistance. This work was supported by the Intramural Program of the NIH, the CIBEREHD Program, and grants to J.C. from Instituto de Salud Carlos III, and cofunded by the European Regional Development Fund (ERDF) (CP13/00160 and PI14/00783), the European Commission MC-CIG (COLONGEVA), the Spanish Association Against Cancer (AECC, GCB13131592CAST), and the Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya (2014 SGR 135 and 2014 SGR 903). Further support was provided by National Service Foundation (NSF) Grant MCB-1517506 to D.C. I.Q., and M.V.-C. received a Formación de Profesorado Universitario (FPU)-fellowship from Ministerio de Educación, Cultura y Deporte; K.T. received a Personal Investigador en Formació from the Universitat Autònoma de Barcelona; A.E. received a Formación de Personal Investigador (FPI) fellowship from Ministerio de Ciencia e Innovación; and Z.Y. received a fellowship from the Scientific and Technological Research Council of Turkey (TUBITAK) 2219 Program. CIBEREHD is funded by the Instituto de Salud Carlos III. The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of 2N and 4N isogenic clones. A) Percentage of FISH signals for 4 locus-specific probes on 5000 parental DLD-1 cells deciphering presence of 4N cellular subpopulation. B) Diagram showing procedure for FACS and generation of pure 2N and 4N isogenic clones. Sort window P5-selected cells have high forward scatter, and are larger and have higher probability of being 4N than cells with low forward scatter. C) Growth curve for DLD-1–derived 2N and 4N clones indicated in different colors. Statistical analysis was performed on trends by R package geepack for longitudinal data analysis. D) Comparison of total DNA, RNA, and protein amounts in seven 2N and seven 4N DLD-1 clones. Each data point represents measurement for biologic replicates (n = 4–9). E, F) Measurements for area (E, μm2) and volume (F, μm3) for ∼5000 DLD-1 cells of each 2N and 4N individual clone are represented. Data are reported as means ± sem. **P < 0.001, ***P < 0.0001.
Figure 2
Figure 2
Gene expression profiling. A) Hierarchical clustering for genes that distinguished between DLD-1 derived 2N and 4N clones. Top-ranked 50 genes sorted by FDR are shown. B) Summary of gene set enrichment analysis showing most overrepresented pathways in 4N DLD-1 cells included in Molecular Signature Database. C) Gene set enrichment analysis for DNA replication and mitosis comparing 2N and 4N clones. D) Immunoblot validating up-regulation of RRM2 and phospho-MCM2 in DLD-1 4N clones. Tubulin was used as protein loading control. E) Densitometry analysis of immunoblot represented as ratio between protein of interest and loading control protein GAPDH. *P < 0.05.
Figure 3
Figure 3
Increased replication stress in 4N cells. A) Immunoblot analysis of total CHK1 and phospho-CHK1 levels in DLD-1 2N and 4N cells. GAPDH was used as protein loading control. B) Immunoblot showing increased levels of both RPA32 and phospho-RPA32 in DLD-1 4N cells. C) Analysis of phospho-CHK1 levels after addition of aphidicolin (0.2 µM) for 24 h to both sets of DLD-1 clones. GAPDH was used as protein loading control. D) Immunoblot analysis of total CHK1 and phospho-CHK1 in RKO 2N and 4N cells. GAPDH was used as protein loading control. E) Immunoblot showing increased levels of phospho-RPA32 vs. RPA32 in RKO tetraploid cells. GAPDH was used as protein loading control. F) Analysis of phospho-CHK1 levels after addition of aphidicolin (0.2 µM) for 24 h to both sets of RKO clones. GAPDH was used as protein loading control. G) Graph depicting percentage of growth impairment of 4N compared to 2N DLD-1 cells as measured by colony formation assay after treatment with ATRi (10 µM) for 24 h (n = 3/clone). H) Immunoblot analysis of total CHK1 and phospho-CHK1 levels in RPE1 wild-type (2N) and posttetraploid (4N) clones. GAPDH was used as protein loading control. I) Immunoblot showing increased levels of phospho-RPA32 vs. RPA32 in posttetraploid RPE1 cells. GAPDH was used as protein loading control. J, K) Plots showing time-course experiments to characterize delay in cell cycle. DLD-1 cells were pulse-labeled with BrdU and analyzed by FACS every 2 h for 12 h period. 4N cells exhibit slower progression through S phase (J) and slower rate going through G2 phase (K). L) Histogram showing mitotic timing of 2N and 4N cells quantified by live cell imaging in DLD-1 (n = 50 cells/clone). M) Histogram showing mitotic index of 2N and 4N DLD-1 cells based on DAPI imaging (n = 50 cells/clone). Statistical analysis was performed on trends using R package geepack for longitudinal data analysis. Data are reported as means ± sem. **P < 0.001, ***P < 0.0001 (n.s., not significant).
Figure 4
Figure 4
Levels of DNA damage in 4N cells. A) Representative images of immunofluorescence for 53BP1 (red) and γ-H2AX (green). DAPI was used for nuclear counterstaining. Scale bar, 5 µm. B) Histogram depicting number of 53BP1 foci per DNA content for 2N and 4N DLD-1 cells (n > 240 cells/clone). C) Histogram showing number of γ-H2AX foci per DNA content for 2N and 4N DLD-1 cells (n > 240 cells/clone). D) Quantification of γ-H2AX foci per DNA content in RKO 2N and 4N clones (n > 100 cells/clone). E) Representative images of DAPI (blue), EdU (red), γ-H2AX (green), and merged images in both 2N and 4N DLD-1 cells. Scale bar, 5 µm. F) Plotted is normalized number of γ-H2AX foci per EdU-positive nuclei in DLD-1 cells (n > 240 cells/clone). G) Quantitative analysis of γ-H2AX foci per mitotic cell for two 2N and two 4N DLD-1 clones. For all these analyses, integrated density of DAPI signal was used to calculate DNA content of each cell. Data are reported as means ± sem. **P < 0.001, ***P < 0.0001.
Figure 5
Figure 5
Assessment of intercellular genetic heterogeneity. A, B) Dot plot depicting number of chromosomes in individual cells from six 2N and six 4N DLD-1 clones (A) and one 2N and two 4N RKO clones (B). Black lines denote modal chromosome number for each clone (n = 100 metaphases/clone). C, D) Graphs illustrate percentage of cells with corresponding FISH signals for 2N (C) and 4N (D) DLD-1 clones after total of 1000 nuclei were analyzed and show higher chromosomal variability in 4N cells. E) Representative FISH images for both sets of clones. FISH panel 1 includes fluorescent-labeled BAC clones for EGFR (7p) and CCND1 (11q), and FISH panel 2 includes fluorescent-labeled BAC clones TERC (3q) and CDX2 (13q). Scale bar, 2 µm. F) Circos plots illustrate comparative frequency of copy number detected by aCGH in early DLD-1–passaged 2N (inner histogram; n = 3) and 4N (outer histogram; n = 12) clones, as well as in late passaged 2N (inner histogram; n = 3) and 4N (outer histogram; n = 5) clones. Human chromosome ideogram is laid out at periphery of circle. Red blocks indicate copy number gains; green blocks, copy number losses. G) Representative image of SKY analysis performed in DLD-1, indicating by arrowhead presence of de novo structural chromosomal aberrations in 4N cells.
Figure 6
Figure 6
Replication stress-associated CIN. A) Examples of mitotic defects in anaphase cells immunostained for kinetochores (green) and microtubules (red). Images show examples of most frequently observed defects, including lagging chromosomes (top row), multiple missegregating chromosomes (middle row), and chromosome bridges and acentric fragments (bottom row). Grayscale images at bottom right corners of each of DNA images are single focal planes of DAPI-stained chromosomes shown for easier visualization of mitotic defects. Arrowheads in grayscale images and merged panels point to specific mitotic defect. Scale bar, 10 µm. B, C) Frequencies of anaphase defects in normal culture conditions for 2N and 4N DLD-1 (B) and RKO (C) cells. D, E) Frequencies of anaphase defects in 2N and 4N DLD-1 (D) and RKO (E) cells treated with aphidicolin (0.2 µM) for 24 h. F, G) Frequencies of anaphase defects in 4N DLD-1 (F) and RKO (G) cells supplemented with mixture of nucleosides (30 and 100 µM, respectively) for 24 h. Data are represented as means ± sem. *P < 0.05, ***P < 0.0001.
Figure 7
Figure 7
Increased migratory capabilities of 4N cells and their prevalence in invasive fronts of colorectal primary tumors. A) Boyden chamber assay representative images showing migration capability of 2N and 4N DLD-1 clones. B) Plotted is mean area of scratch closure resulting from wound-healing assay at 24 and 48 h after scratching for both early (passages 5–10) and late (passages 25–30) passaged 2N and 4N clones (P < 0.05 and P < 0.0001, respectively). C) Bar graph showing quantification of Matrigel invasiveness for 2N and 4N cells. Data are represented as mean number of invasive cells ± sem (n = 3). *P < 0.05. D) Representation of centrosome number in migrating cells during wound-healing assay. Quantification was performed by coimmunostaining of γ-tubulin and cyclin D1 (n = 250–350). E) Microscopic assessment of FISH signals using fluorescent probes for centromeres corresponding to chromosomes 4 (FITC) and 6 (Cy3) in colorectal primary tumor (top) and its associated invasive front (bottom). Single isolated nuclei are outlined with dashed circles. All 3 nuclei from primary tumor display 2 signals per probe, whereas 3 or 4 signals per probe were detected in nuclei from invasive front. F) Distribution of centromere probes signals for chromosome 4 and chromosome 6 in 9 colorectal primary tumors and their corresponding invasive fronts (P < 0.0001). G) Percentage of cells with extra centrosomes (>2 centrosomes per cell) in colorectal primary tumor samples and their corresponding invasive fronts. *P < 0.05.
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