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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.

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).

Hierarchical clustering of cell surface markers in HCT116 and RPE1 trisomic cell lines. The expression of 49 cell surface markers (each column represents a single protein) in tri- and tetrasomic clones derived from HCT116 (A) and RPE1 (B) parental cell lines - each row represents a different clone, namely HCT116 3/3 (trisomy 3 clones 11 and 13), 5/3 (trisomy 5), 8/3 (trisomy 8 clones 3, 5 and 7), 13/3 (trisomy 13 clones 2 and 3), 18/3 (trisomy 18 clones 1 and 2), 21/3 (trisomy 21 clones 1 and 3), 5/3 3/3 (trisomy 5 and 3), 5/4 (tetrasomy 5) and RPE1 3/3 (trisomy 3 clone 1 and 2), 5/3 (trisomy 5 clone 3 and 7), 12/3 (trisomy 12), 5/3 12/3 (trisomy 5 and 12), 21/3 (trisomy 21) - are 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.

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).

Knockdown efficiency of the candidates by siRNA and its effect on cell proliferation and cell death. (A) Expression intensities of CD73, CD230, CD47, CD49b, CD55 and CD95 after knockdown using siRNAs (red lines), as well as both not transfected (black line) and non-targeting siRNA (grey line) controls, in HCT116 parental and HPT1 post-tetraploid cells, analyzed at the cell surface level by flow cytometry. (B) Effect of CD73, CD230, CD47 and CD49b siRNA knockdown on cell death, analyzed by % of AnnexinV+ cells. Representative AnnexinV-A vs DAPI-A dot plots are shown for both HCT116 parental and HPT1 post-tetraploid cells. (C) Effect on cell proliferation evaluated by % of cells which incorporated EdU after siRNA knockdown in comparison to not transfected and non-targeting siRNA controls; bar graphs represent the mean �U+ cells (and standard deviations) for three replicates on both HCT116 parental (black bars) and HPT1 (grey bars) cells (no statistically significant differences).

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.

Description of the Supplementary Files

Effect of oxidative stress on CD73, CD230, CD47, CD49b, CD55 and CD95 expression in parental and aneuploid cell lines. Ratios of H2O2-treated cells (100µM 48h) and control cells are shown for the mean fluorescence intensity values (black bars) and percentage of positive cells (grey bars).

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/PrPC 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/PrPC 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. Cancer Res; 77(11); 2914–26. ©2017 AACR.

Deregulation of the G1-S transition of the cell cycle is a common feature of human cancer. Tumor-associated alterations in this process frequently affect cyclin-dependent kinases (Cdk), their regulators (cyclins, INK4 inhibitors, or p27Kip1), and their substrates (retinoblastoma protein). Although these proteins are generally thought to act in a linear pathway, mutations in different components frequently cooperate in tumor development. Using gene-targeted mouse models, we report in this article that Cdk4 resistance to INK4 inhibitors, due to the Cdk4 R24C mutation, strongly cooperates with p27Kip1 deficiency in tumor development. No such cooperation is observed between Cdk4 R24C and p18INK4c absence, suggesting that the only function of p18INK4c is inhibiting Cdk4 in this model. Cdk4R/R knock in mice, which express the Cdk4 R24C mutant protein, develop pituitary tumors with complete penetrance and short latency in a p27Kip1−/− or p27Kip1+/− background. We have investigated whether this tumor model could be useful to assess the therapeutic activity of cell cycle inhibitors. We show here that exposure to flavopiridol, a wide-spectrum Cdk inhibitor, significantly delays tumor progression and leads to tumor-free survival in a significant percentage of treated mice. These data suggest that genetically engineered tumor models involving key cell cycle regulators are a valuable tool to evaluate drugs with potential therapeutic benefit in human cancer.

Supplementary Table 1 from Cooperation between Cdk4 and p27kip1 in Tumor Development: A Preclinical Model to Evaluate Cell Cycle Inhibitors with Therapeutic Activity

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

Cell cycle progression requires the co-ordinated activation of several kinases, some of which are activated upon the binding of a cyclin subunit. At least four of these so-called cyclin-dependent kinases, namely Cdk4, Cdk6, Cdk2 and Cdk1, have specific roles at particular stages of the cell cycle, including passage through the various cell cycle transitions and the response to specific checkpoints. Not surprisingly, most human tumors carry mutations that deregulate at least one of these kinases. To analyze their specific role in vivo, we are generating strains of gene-targeted mice carrying either activated or defective alleles of these Cdks. As an example, Cdk4 expression appears to be expendable in most cell types since mice lacking Cdk4 are viable. Yet, Cdk4 mutant mice are smaller in size and infertile (only partial infertility in males). In addition, Cdk4 defective mice develop insulin dependent diabetes early in life. However, the importance of these Cdks in tumor cell cycles is underscored by the phenotype of knock in mice where the normal Cdk4 gene has been replaced by a Cdk4 R24C (insensitive to INK inhibitors) mutant. These animals develop a wide spectrum of spontaneous tumors and are highly susceptible to specific carcinogenic treatments. These models are being used now to understand how deregulation of these Cdks leads to cancer development and will be a valuable tool to design and validate new therapeutic strategies against tumour development.