Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals

Abstract

Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.

Keywords: Plk4; aneuploidy; centriole; centrosome amplification; genomic instability; mitosis; tumorigenesis.

Figure 1. related to Figure S1. A modest increase in Plk4 promotes centrosome amplification and aneuploidy in vitro

(A) System used for doxycycline-inducible expression of Plk4. (B) Quantification of the level of centrosomal Plk4 in Plk4^Dox MEFs. N = 3, >150 centrosomes per experiment. (C) Quantification of the level of centrosome amplification in Plk4^Dox MEFs. N = 3, >150 cells per experiment. (D) Immunofluorescent images of centrosomes in Plk4^Dox MEFs. (E) Quantification of anaphase phenotypes in Plk4^Dox MEFs. N = 3, >150 cells per experiment. (F) Quantification of anaphase lagging chromosomes in Plk4^Dox MEFs. N = 3, >150 cells per experiment. (G) Quantification of the fraction of cells having <2 or >2 copies of chromosome 15 or 16. N = 3, >150 cells per experiment. (H) Immunofluorescent images of FISH performed on Plk4^Dox MEFs using probes against chromosome 15 and 16. Arrowheads mark each copy of Chr15 or Chr16. (I) Graph showing the fold increase in cell number for Plk4^Dox MEFs. N = 3, performed in triplicate. (J) Graph showing the fold increase in cell number for Plk4^Dox MEFs expressing SpCas9 and an sgRNA against p53. N = 3, performed in triplicate. All data represent the means ±SEM. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001; two-tailed Student’s t-test. Scale bars represent 10 μm.

Figure 2. related to Figure S2 and S3. Increased Plk4 levels promote chronic centrosome amplification in multiple tissues

(A) Fold increase in Plk4 mRNA in tissues from Plk4^Dox mice treated with doxycycline for 1 month. N = 3, performed in triplicate. (B and C) Quantification of the level of centrosome amplification in tissues from Plk4^Dox mice treated with doxycycline for 1 or 8 month. N ≥ 4. (D) Quantification of centrosome number in tissues from Plk4^Dox mice treated with doxycycline for 8 months. N = 4. (E) Representative images of centrosomes in tissues from doxycycline treated Plk4^Dox or control animals. (F–H) Quantification of the fraction of cleaved caspase 3, TUNEL, or Ki67 positive cells in tissues from Plk4^Dox mice treated with doxycycline for 1 month. Data are means ±SEM (N = ≥4). All data represent the means ±SEM. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001; two-tailed Student’s t-test. Scale bars represent 10 μm.

Figure 3. related to Figure S4. Centrosome amplification drives aneuploidy in vivo

(A) Proportion of severely aneuploid (4N ± > 2 chromosomes) splenocytes from control and Plk4^Dox mice treated with doxycycline for 1 or 8 months. N = 3, > 120 cells per experiment. (B) Table shows the fraction of aneuploid cells determined by single cell sequencing of epidermal cells from doxycycline-treated control or Plk4^Dox mice. (C) Genome-wide copy number plots of aneuploid single cells sequenced from the epidermis of 3 Plk4^Dox mice treated with doxycycline for 12–18.5 months. Individual cells are represented in rows with copy number states indicated in colors. All data represent the means ±SEM. ^*P < 0.05; two-tailed Student’s t-test. Scale bars represent 10 μm.

Figure 4. related to Figure S4. Centrosome amplification promotes tumor initiation

(A) Quantification of the level of centrosome amplification in APC^Min/+; Plk4^Dox MEFs. N = 3, >150 cells per experiment. (B) Quantification of anaphase lagging chromosomes in APC^Min/+; Plk4^Dox MEFs. N = 3, >84 cells per experiment. Scale bar represents 10 μm. (C) Frequency of micronuclei observed in APC^Min/+; Plk4^Dox MEFs. N = 3, >50 cells per experiment. (D and E) APC^Min and APC^Min/+; Plk4^Dox mice were treated with doxycycline from 10 days of age and sacrificed at 90 days old. Quantification shows the level of centrosome amplification in the intestines or intestinal polyps of APC^Min and APC^Min/+; Plk4^Dox mice. N = 3, >150 cells per experiment. (F) (Left) Immunofluorescence staining of an intestinal polyp and (Right) a magnified view of centrosomes in this tumor. Scale bars represent 200 μm (left) and 10μm (right). (G and H) Quantification of tumor number (G) or size (H) in 90 day old APC^Min and APC^Min/+; Plk4^Dox mice. (I) Images show intestinal polyps in an APC^Min and APC^Min/+; Plk4^Dox mouse. All data represent the means ±SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and NS (not significant) indicates P > 0.05; two-tailed Student’s t-test.

Figure 5. related to Figure S5. Centrosome amplification promotes spontaneous tumorigenesis

(A) Kaplan-Meier survival analysis of Plk4^Dox and control (C57BL/6J) mice chronically fed doxycycline from 1–2 months of age. P value was calculated using the Log-rank test. (B) Quantification of the level of centrosome amplification in tumors from Plk4^Dox and p53^−/− mice. Horizontal lines represent the mean and bars represent ±SEM. (C) Representative examples of the different tumor types that develop in doxycycline-treated Plk4^Dox mice. (D and E) GISTIC analysis of low-coverage whole-genome sequencing of Squamous Cell Carcinomas (SCCs) and lymphomas from doxycycline-treated Plk4^Dox mice shows gains of specific chromosomes. Scale represents Q values. (F) Low-coverage whole-genome sequencing (WGS) plots for a Sarcoma and a Squamous Cell Carcinoma derived from Plk4^Dox mice. (G) (Top) WGS plots from a T-Cell Lymphoma derived from doxycycline-treated Plk4^Dox mice. (Bottom) Genome-wide copy number plots of aneuploid single cells sequenced from the same T-cell lymphoma. 12/39 sequenced cells showed evidence of aneuploidy. Individual cells are represented in rows with copy number indicated in colors. (H) (Top) WGS plots from a B-Cell Lymphoma derived from doxycycline-treated Plk4^Dox mice. (Bottom) Genome-wide copy number plots of aneuploid single cells sequenced from the same B-cell lymphoma. 32/47 sequenced cells showed evidence of aneuploidy.