LAUSR

Simple Summary CDC20 has been shown to function as an oncogene in multiple human malignancies and is recognized as a promising target for developing novel therapeutic strategies. Previous studies showed the role of CDC20 in cancer development and linked its function to drug resistance. Drug resistance can be acquired by multiple mechanisms, including chromatin organization and dynamic changes. It was previously reported that cancer cells with enlarged nuclei and diffuse chromatin showed higher resistance to anti-cancer therapy. We showed that CDC20 interacts with hnRNPU in the chromatin fraction and regulates its ubiquitination in breast cancer cells. CDC20-mediated hnRNPU ubiquitination modulates chromatin condensation by regulating the formation of the CTCF–cohesin complex. Dysregulation of the CDC20–hnRNPU axis results in diffuse chromatin and leads to drug resistance in breast cancer cells. Our findings shared new insights into the implication of the CDC20–hnRNPU axis in regulating chromatin organization and drug resistance, suggesting that the CDC20–hnRNPU axis could be a good target for cancer therapy. Abstract Cell division cycle 20 (CDC20) functions as a critical cell cycle regulator. It plays an important role in cancer development and drug resistance. However, the molecular mechanisms by which CDC20 regulates cellular drug response remain poorly understood. Chromatin-associated CDC20 interactome in breast cancer cells was analyzed by using affinity purification coupled with mass spectrometry. hnRNPU as a CDC20 binding partner was validated by co-immunoprecipitation and immunostaining. The molecular domain, comprising amino acid residues 461–653, on hnRNPU required for its interaction with CDC20 was identified by mapping of interactions. Co-immunoprecipitation showed that CDC20-mediated hnRNPU ubiquitination promotes its interaction with the CTCF and cohesin complex. The effects of CDC20–hnRNPU on nuclear size and chromatin condensation were investigated by analyzing DAPI and H2B-mCherry staining, respectively. The role of CDC20–hnRNPU in tumor progression and drug resistance was examined by CCK-8 cell survival and clonogenic assays. Our study indicates that CDC20-mediated ubiquitination of hnRNPU modulates chromatin condensation by regulating the interaction between hnRNPU and the CTCF–cohesin complex. Dysregulation of the CDC20–hnRNPU axis contributes to tumor progression and drug resistance.