Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes

Abstract

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

Keywords: DNA replication stress; alternative lengthening of telomeres (ALT); chromosomal instability in neoplasia (CIN); intratumor genomic heterogeneity; polyploidy; therapy resistance; whole genome doubling (WGD).

Figure 1

Karyotypic evolution in the U2-OS osteosarcoma cell line. (A) Representative, pseudo-colored, multicolor-FISH karyotypes of ten major subclones (Sc), from five U2-OS-derivative cell lines (SL) composed of 69–80 chromosomes. In U2-OS cells, every homologue of the human karyotype is affected by clonal structural or numerical aberrations. Despite the karyotypic diversity, a monoclonal origin of all side lines is evident by the common presence of six identical recombinant chromosomes (pink arrows). Based on the constitution of chromosome 5, the WT1 cells probably represent the most ancestral population. The CDT1-overexpressing, and Doxorubicin-resistant R1 and R2 cells derive from WT2. White arrows depict clone-specific rearrangements. Note that several evolutionary steps (i.e., the process from WT1 to WT2 Sc1-2 or the evolution of CDT1 and R1 cells) were accompanied by karyotypes bearing multiple duplicated copies of identical clonal recombinant chromosomes (asterisks) suggesting that leaps in karyotypic evolution were accompanied by whole genome duplication (WGD) followed by multiple chromosome losses. (B) Jumping translocations of large recombinant segments clonally present in most U2-OS-derivative cell lines and subclones. (C) Chromoanagenesis was recorded only in one recombinant chromosome, composed of multiple alternate segments of chromosomes 5 and 19, in the chemo-resistant R2 cells. (D) Identical large genome imbalances identified by aCGH are stably maintained between the U2-OS-derivative cell lines despite the karyotypic alterations produced by extensive chromosome breakage and illegitimate rejoining. Red circles indicate the presence of the same imbalance in all 5 cell lines, pink circles depict undistinguishable duplications/deletions in 4 out of 5 karyotypically-diverse U2-OS cell lines. Lower boxes include partial karyograms involving genomic material of the chromosome analyzed by aCGH, representing two major subclones per side line.

Figure 2

Distribution of random structural chromosomal instability in neoplasia (CIN) among co-dividing alternative lengthening of telomeres (ALT) cells and the possible role of WGD in the perpetuation of the integrity of driver chromosomal rearrangements. (A) Uneven distribution of non-clonal structural chromosomal rearrangements between 15 randomly picked, co-dividing cells from 4 human ALT cell lines. Bars indicate structural CIN load, calculated as breakpoints/chromosome. Chromosome number/metaphase is indicated above each data bar. From every cell line we selected 3 cells with the lower rates of structural chromosomal instability. Interestingly, half of the 12 CIN escapee cells (red arrows and numbers) were byproducts of WGD or showed evidence of WGD reduction. (B) Pseudo-colored partial karyograms of two co-dividing VA-13 mitotic nuclei belonging to the major clones and each bearing 69 chromosomes. In contrast to #13, cell #12 displays extremely low rates of structural CIN and presents several duplicated copies of clonal recombinant chromosomes, suggesting WGD followed by multiple chromosome losses.

Figure 3

Polyploidization protects the abnormal cancer genome from ongoing structural chromosome aberrations and promotes intratumor heterogeneity. (A) Karyotypic analysis in two groups of 15 VA-13 cells, harvested 10 days after exposure to gamma irradiation, reveals significantly lower rates of random structural CIN in the cells that have undergone 1 or 2 rounds of WGD (composed from 104–178 chromosomes) as compared to those undergoing mitosis of the major VA-13 clones (composing of 64–78 chromosomes). Structural CIN was calculated as breakpoints/chromosome/cell. (B) Similar results were obtained in the osteosarcoma Saos-2 cells suffering from DNA replication stress upon prolonged p21 overexpression that duplicates the average structural CIN load. (C) Distribution of chromosome counts in 50 co-dividing VA-13 cells harvested in subsequent passages after 2.4 Gy of gamma irradiation (1 passage = 2–4 days in culture). Red dotted line represents chromosome numbers proximal to major clones. Pink boxes include cells that underwent one round of WGD. Blue boxes include cells that underwent more than one round of WGD. Note that despite the decline in the rates of WGD by time in culture, very few intermediate chromosome counts were recorded between the distinct ploidy indices, suggesting that cells with heavily unbalanced DNA content are less proficient at dividing. (D) M-FISH pseudo colored partial karyotypes of two WGD clones, generated with p21-overexpression-mediated genome reshuffling, display duplicated copies of novel complex recombinant chromosomes (arrows) not observed in non-endoreduplicated cells, suggesting that WGD increases tolerance of novel structural aberrations and thus contributes to genomic heterogeneity.