Experimental Approaches to Generate and Isolate Human Tetraploid Cells.

Cancer cells are frequently affected by large-scale chromosome copy number changes, such as polyploidy or whole chromosome aneuploidy, and thus understanding the consequences of these changes is important for cancer research. In the past, it has been difficult to study the consequences of large-scale genomic changes, especially in pure isogenic populations. Here, we describe two methods to generate tetraploid cells induced either by cytokinesis failure or mitotic slippage. These treatments result in mixed population of diploids and tetraploids that can be analyzed directly. Alternatively, tetraploid populations can be established by single cell clone selection or by fluorescence activated cell sorting. These methods enable to analyze and compare the consequences of whole-genome doubling between the parental cell line, freshly arising tetraploid cells, and post-tetraploid aneuploid clones.

Genetic instability from a single S phase after whole-genome duplication.

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.

Loss of USP28 and SPINT2 expression promotes cancer cell survival after whole genome doubling.

BACKGROUND: Whole genome doubling is a frequent event during cancer evolution and shapes the cancer genome due to the occurrence of chromosomal instability. Yet, erroneously arising human tetraploid cells usually do not proliferate due to p53 activation that leads to CDKN1A expression, cell cycle arrest, senescence and/or apoptosis. METHODS: To uncover the barriers that block the proliferation of tetraploids, we performed a RNAi mediated genome-wide screen in a human colorectal cancer cell line (HCT116). RESULTS: We identified 140 genes whose depletion improved the survival of tetraploid cells and characterized in depth two of them: SPINT2 and USP28. We found that SPINT2 is a general regulator of CDKN1A transcription via histone acetylation. Using mass spectrometry and immunoprecipitation, we found that USP28 interacts with NuMA1 and affects centrosome clustering. Tetraploid cells accumulate DNA damage and loss of USP28 reduces checkpoint activation, thus facilitating their proliferation. CONCLUSIONS: Our results indicate three aspects that contribute to the survival of tetraploid cells: (i) increased mitogenic signaling and reduced expression of cell cycle inhibitors, (ii) the ability to establish functional bipolar spindles and (iii) reduced DNA damage signaling.