Up to 80% of human cancers, in particular solid tumors, contain cells with abnormal chromosomal numbers, or aneuploidy, which is often linked with marked chromosomal instability. Whereas in some tumors the aneuploidy occurs by missegregation of one or a few chromosomes, aneuploidy can also arise during proliferation of inherently unstable tetraploid cells generated by whole genome doubling from diploid cells. Recent findings from cancer genome sequencing projects suggest that nearly 40% of tumors underwent whole genome doubling at some point of tumorigenesis, yet its contribution to cancer phenotypes and benefits for malignant growth remain unclear. Here, we investigated the consequences of a whole genome doubling in both cancerous and non-transformed p53 positive human cells. SNP array analysis and multicolor karyotyping revealed that induced whole-genome doubling led to variable aneuploidy. We found that chromosomal instability (CIN) is a frequent, but not a default outcome of whole genome doubling. The CIN phenotypes were accompanied by increased tolerance to mitotic errors that was mediated by suppression of the p53 signaling. Additionally, the expression of pro-apoptotic factors, such as iASPP and cIAP2, was downregulated. Furthermore, we found that whole genome doubling promotes resistance to a broad spectrum of chemotherapeutic drugs and stimulates anchorage-independent growth even in non-transformed p53-positive human cells. Taken together, whole genome doubling provides multifaceted benefits for malignant growth. Our findings provide new insight why genome-doubling promotes tumorigenesis and correlates with poor survival in cancer.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrP and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73 cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrP was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. .

<div>Abstract<p>Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrP^C and the immunosuppressive cell surface enzyme ecto-5′-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73⁺ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrP^C was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. <i>Cancer Res; 77(11); 2914–26. ©2017 AACR</i>.</p></div>

<p>Description of the Supplementary Files</p>

<p>Supplementary Tables S1-S3. Detailed description of antibodies used for the cell surface screenings performed with the initial 300 antibody high-throughput screening panel (S1), the 49-marker validation screening panel (S2), and the 36-marker mouse tumor screening panel (S3). Supplementary Table S4. Mean fluorescence intensity and percentage of positive cells for each surface marker staining across the cell lines initially analyzed with the MACS® Marker Screening panel. Supplementary Table S5. Mean fluorescence intensity and percentage of positive cells for the 49 surface markers selected for validation across all the cell lines, grouped according to the karyotype and normalized with the corresponding parental cells. Supplementary Tables S6. Aneuploid-to-diploid ratios obtained from previously published proteome data (7) for the proteins analyzed in this study. Supplementary Table S7. Immunophenotypic characterization of mouse models of aneuploidy (breast tumors, n=7) with a 29-marker screening panel, as well as normal mammary gland (control; n=2).</p>

<p>Effect of ER stress on CD73, CD230, CD47, CD49b, CD55 and CD95 expression in parental and aneuploid cell lines. Ratios of cells treated with multiple ER stress inducers - glucose-deprivation (black bars), brefeldin A (BFA; red bars) 1ug/mL, thapsigargin (Thaps; blue bars) 0.25ug/ml or tunicamycin (TuniM; green bars) 2.5ug/mL for 48h - and control cells are shown for the mean fluorescence intensity values (top graph) and percentage of positive cells (bottom graph).</p>

<p>Hierarchical clustering analysis of multiple cell surface markers in aneuploid cell lines. The expression of 291 cell surface markers (each row represents a single protein) in 5 different aneuploid cell lines (each column represents a different cell line, namely RPE1 5/3 12/3 - trisomy of chromosome 5 and 12 -, RPE1 21/3 - trisomy of chromosome 21 -, HCT116 5/3 - trisomy of chromosome 5 -, RPT1 - RPE1 post-tetraploid clone -, HPT1 - HCT116 post-tetraploid clone -) is shown in a matrix format. The values were normalized to the corresponding parental cell line and are represented by a color scale where red and green colors indicate values of increased- and decreased-expression, respectively.</p>

<p>Expression of CD230 upon serum-starvation in parental and trisomic cells. Representative dot plots of FSC-A vs CD230-A for HCT116 parental and HCT116 trisomic clones - 18/3 cl2 and 21/3 cl1 and 3 - cultured under normal conditions (10% FBS, control) or under serum-starvation (0% FBS) for 3 days, are shown.</p>