Project Summary Cancer is characterized by genomic, epigenetic, and metabolic changes, but also by a failure in cell/nuclear size control. Nevertheless, little is known about size regulation in cancer cells and whether failures in size control contribute to tumorigenesis. Whole genome doubling (WGD), which results in tetraploidy and is a frequent intermediate event in tumor progression, is a key process that can alter cell and nuclear size. However, while ploidy and nuclear size tend to scale proportionally in normal human cells, DNA content and nuclear size do not always correlate in cancer cells. This suggests that understanding how nuclear size is controlled following changes in DNA content may uncover important mechanisms underlying tumorigenesis. Addressing this issue requires a suitable experimental model, such as the one that will be used for this project. This model consists of a panel of tetraploid (4N) clonal cell lines that were derived by inducing WGD in colorectal and breast cancer cells and can be divided into two groups (small and large) based on nuclear size. Previous studies showed that the small and large 4N DLD1 clones display distinct mitotic phenotypes, which raised important questions about the mechanisms of size control in 4N cells and the impact of nuclear size on other cell phenotypes. Indeed, preliminary results indicate that the small 4N clones have increased tumor-like behavior in vitro, outperforming the large 4N clones and 2N parental cells in soft agar colony formation assays. These size-specific phenotypic differences suggest that the small 4N clones may also be more tumorigenic in vivo than the large 4N clones due to their ability to restrict nuclear size scaling after WGD. This hypothesis will be tested in a first research aim using a combination of in vitro, in vivo, and in silico methods and two experimental models, including the novel panel of small and large 4N clones derived from 2N DLD1 cells (for a running total of 16 cell lines) and genomic, histopathology, and clinical data from The Cancer Genome Atlas (TCGA). A second research aim will test the hypothesis that the nuclear volumes of the 4N clones are dictated by the degree of genomic compaction and will identify the transcriptional and epigenetic mechanisms that regulate nuclear size after WGD in colon- and breast- derived cells. These experiments will address a fundamental yet underexplored question of whether variations in nuclear size have important functional consequences that contribute to tumor formation following WGD. The findings of this project could be leveraged for cancer diagnostics and unveil new cancer therapeutic targets. This project will provide ample training opportunities in the use of animal models, computational analysis, and mass spectrometry from an interdisciplinary group of leading researchers to help the applicant become an independent scientist. The intellectual environment, resources, and training programs provided to the app...