A role for polyploidy in the tumorigenicity of Pim-1-expressing human prostate and mammary epithelial cells

Abstract

Background: Polyploidy is a prominent feature of many human cancers, and it has long been hypothesized that polyploidy may contribute to tumorigenesis by promoting genomic instability. In this study, we investigated whether polyploidy per se induced by a relevant oncogene can promote genomic instability and tumorigenicity in human epithelial cells.

Principal findings: When the oncogenic serine-threonine kinase Pim-1 is overexpressed in immortalized, non-tumorigenic human prostate and mammary epithelial cells, these cells gradually converted to polyploidy and became tumorigenic. To assess the contribution of polyploidy to tumorigenicity, we obtained sorted, matched populations of diploid and polyploid cells expressing equivalent levels of the Pim-1 protein. Spectral karyotyping revealed evidence of emerging numerical and structural chromosomal abnormalities in polyploid cells, supporting the proposition that polyploidy promotes chromosomal instability. Polyploid cells displayed an intact p53/p21 pathway, indicating that the viability of polyploid cells in this system is not dependent on the inactivation of the p53 signaling pathway. Remarkably, only the sorted polyploid cells were tumorigenic in vitro and in vivo.

Conclusions: Our results support the notion that polyploidy can promote chromosomal instability and the initiation of tumorigenesis in human epithelial cells.

Figure 1. Late passage, polyploid, Pim-1-expressing RWPE1 prostate cells are tumorigenic in nude mice.

(A) Western blotting for Pim-1 and actin in RWPE1 cells overexpressing Pim-1 or vector control (Neo). Early and late passage cells are shown. (B) Cell cycle profiles of Pim-1 overexpressing RWPE1 cells shows polyploidy in late passage cells. (C) FISH analysis of late passage Pim-1 overexpressing RWPE1 cells using probes for chromosomes 13 and 21 show chromosome doubling. (D) Sample images of H&E stained sections from RWPE1 xenografts. Only late passage Pim-1 expressing cells were tumorigenic.

Figure 2. Isolation of matched diploid/polyploid RWPE1-Pim-1 cells by cell sorting.

(A) Diagram showing scheme for isolation of diploid (2N) and polyploid (>4N) Pim-1 cells by FACS sorting based on DNA content. Bottom panel is the FACS profile of sorted cells after several passages to get enough cells for FACS analysis. (B) Western blotting of Pim-1 and other markers in FACS sorted cells shows similar expression levels in sorted diploid and polyploid cells. (C) Cell counting of diploid and polyploid Pim-1 overexpressing cells. (D) Western blotting shows that the p53 pathway is intact in all FACS-sorted RWPE1 cells as demonstrated by the induction of p53 and p21 following daunorubicin treatment.

Figure 3. Chromosomal abnormalities in sorted polyploid RWPE1-Pim-1 cells.

(A) Percentage of cells showing total chromosome number, numerical and structural aberrations from FACS-sorted RWPE1 cells. Total 31 to 40 metaphase spreads were analyzed per sample and scored for the chromosome number, numerical aberrations, and structural aberrations. (B) Representative SKY images of sorted Neo, diploid and polyploid RWPE1-Pim-1 cells. Neo: 49,X,del(Y),+5, t(9;20;X),t(11;18),+12,del(12),t(12;21),t(14;18),+15,t(14;18),+20,t(18;20). Diploid: 49,X,del(Y),t(3;4),+5,+5,t(9;20;9),+12,t(5;12)(12;21),t(14;18),t(7;18),+20,t(17;18;20). Polyploid: 97,XX,del(Y),+1,+1,+1,t(1;5),+2,+2,+3,+3,+3,t(3;7),+4,+4,+5,+5,+5,+5,+5,+5,t(5;21),+6,+6,+7,+8,+8,+9,+9,t(9;20;9)×2,+10,+10,+11,+11,+12,+12,+12,+12,t(5;12)×2(12;21)×2,+13,+13,+14,+14,t(14;18)×2,+15,+15,+16,+16,+17,+17,+18,+18,t(7;18)×2,+19,+19,+20,+20,+20,+20,t(17;18;20)×2,+21,+22. (C) Table showing structural chromosomal aberrations. The numbers of derivative chromosomes observed are shown. Polyploid-specific abnormalities are shown in bold.

Figure 4. Polyploidy promotes the tumorigenicity of RWPE1 prostate cells in vitro and in vivo.

(A) Soft agar assay with colony counting in FACS-sorted RWPE1 cells. Colonies larger than 0.5 mm in diameter were counted. Results represent average of triplicate experiments. *p<0.05. (B) Histological analysis of grafts of diploid and polyploid RWPE1-Pim-1 cells recombined with rat UGM and grafted under the kidney capsule. Grafts from polyploid cells contained foci of carcinoma-in-situ with loss of the basal cell marker p63 and high rates of mitotic cells (p-HH3, phospho-histone H3) while diploid cells formed largely small benign looking glands that express p63 and low rates of mitosis (p-HH3). A human-specific Ku70 antibody was used to confirm the human origin of glands. All images were taken at the same magnification of 4×.

Figure 5. FACS sorted, Polyploid, Pim-1 expressing telomerase-immortalized mammary epithelial cells (hTERT-HME1) are tumorigenic in vitro.

(A) FACS profile after cell sorting. hTERT-HME1 cells were sorted based on DNA content. (B) Western blotting of Pim-1 and other markers in sorted cells. Pim-1 expression levels are similar in diploid and polyploid cells. (C) Cell counting of Neo control, diploid and polyploid Pim-1 overexpressing cells. (D) Western blotting for p21 and p53 after daunorubicin treatment shows that p53 function is intact in all FACS-sorted hTERT-HME1 cells.

Figure 6. Chromosomal abnormalities in polyploid hTERT-HME1 cells.

(A) Percentage of cells showing total chromosome number, numerical and structural aberrations from FACS-sorted hTERT-HME1 cells. Note that karyotypes of polyploid and soft agar derived cells are highly heterogeneous, whereas karyotypes of control Neo and diploid cells are more uniform. Total 13 to 40 metaphase spreads were analyzed per sample and scored for the chromosome number, numerical aberrations, and structural aberrations. (B) Representative SKY images from each FACS-sorted hTERT-HME1 cells. Neo: 45,XX,t(6;17),−17. Diploid: 46,XX,del(5),t(5;8). Polyploid: 89,XXXX,+1,+1,t(1;2),+2,+3,+3,+4,+5,+5,+5,del(5),+6,+6,+7,+7,+8,+8,+9,+9,+10,+10,+11,+11,del(11),+12,+12,+13,+13,+14,+15,+15,+16,+16,+17,+17,t(15;17),+18,+18,+19,+19,+20,+20,+21,+21,+22. Soft agar: 89,XXX,+1,+1,t(1;2),+2,+3,+3,+4,+5,+5,+5,del(5), +6,+6,+7,+7,+8,+8,+8,t(8;20),+9,+9,+10,+10,+11,+11,del(11),+12,+12,+13,+13,+14,+14,+15,+16,+16,+17,+17,+18,+18,+19,+19,+20,+21,+21,+22,+22. (C) Tables showing derivative chromosomes in the different sorted cell types. The numbers of derivative chromosomes observed are shown. Polyploid and soft agar derived cell-specific abnormalities are shown in bold.

Figure 7. Polyploidy promotes the tumorigenicity of hTERT-HME1 cells.

Soft agar assay of FACS-sorted diploid and polyploid hTERT-HME1 Pim-1 overexpressing cells and control Neo cells. Note the larger colonies in polyploid cells. Colonies larger than 1 mm in diameter were counted from triplicate of 60 mm dishes. Original magnification: 10×. (B) We isolated cells from soft agar colonies formed by polyploid cells shown in Fig. 7A. The tumorigenic potential of these soft agar-derived polyploid cells was compared side by side with that of the parental polyploid cells by soft agar assay. The soft agar-derived cells formed larger size colonies with higher frequency than the parental polyploid cell population. Original magnification; 4×.