A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

Colorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays provided information of copy number neutral loss of heterozygosity, thus suggesting the occurrence of somatic uniparental disomy (UPD) and uniparental polysomy (UPP) events. The aim of this study is to establish an integrative profiling of recurrent UPDs/UPPs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD/UPP mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs/UPPs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. A meta-analysis using over 300 samples from The Cancer Genome Atlas confirmed our findings. Several regions affected by recurrent UPDs/UPPs contain well-known TSGs, as well as novel candidates such as ARID1A, DLC1, TCF7L2 and DMBT1. In addition, VCAN, FLT4, SFRP1 and GAS7 were also frequently involved in regions of UPD/UPP and displayed high levels of methylation. Finally, sequencing and fluorescence in situ hybridization analysis of the gene APC underlined that a somatic UPD event might represent the second hit to achieve biallelic inactivation of this TSG in colorectal tumors. In summary, our data define a profile of somatic UPDs/UPPs in sporadic CRC and highlights the importance of these events as a mechanism to achieve the inactivation of TSGs.

Somatically acquired uniparental disomies (aUPDs) are frequent events in solid tumors and have been associated with cancer‐related genes. Studies assessing their functional consequences across several cancer types are therefore necessary. Here, we aimed at integrating aUPD profiles with the mutational status of cancer‐related genes in a tumor‐type specific manner. Using TCGA datasets for 1,032 gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), we show a non‐random distribution of aUPD, suggesting the existence of a cancer‐specific landscape of aUPD events. Our analysis indicates that aUPD acts as a “second hit” in Knudson's model in order to achieve biallelic inactivation of tumor suppressor genes. In particular, APC , ARID1A and NOTCH1 were recurrently inactivated by the presence of homozygous mutation as a consequence of aUPD in COAD and READ, STAD and ESCC, respectively. Furthermore, while TP53 showed inactivation caused by aUPD at chromosome arm 17p across all tumor types, copy number losses at this genomic position were also frequent. By experimental and computationally inferring genome ploidy, we demonstrate that an increased number of aUPD events, both affecting the whole chromosome or segments of it, were present in highly aneuploid genomes compared to near‐diploid tumors. Finally, the presence of mosaic UPD was detected at a higher frequency in DNA extracted from peripheral blood lymphocytes of patients with colorectal cancer compared to healthy individuals. In summary, our study defines specific profiles of aUPD in gastrointestinal cancers and provides unequivocal evidence of their relevance in cancer.