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siRNA high-content screening of 3D cultured MCF10A spheroids probing breast tumorigenesis

Waschow, Marcel

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Genome instability and its resulting phenotypes has long been studied and found to be a key factor for cancer development and treatment success. One of the key processes is chromosomal instability (CIN), which results in aneuploidy and is a recurring property of cancer cells. It is considered to be a main contributor to tumor heterogeneity. Breast cancer is the most common form of cancer in women today and shows aneuploidy in approximately 75 % of all cases. Forms with a lower association to aneuploidy, e.g. the luminal subtypes generally have a better prognosis when compared to basal-like or triple-negative ones, which frequently show high rates of aneuploidy. The precursor forms of breast cancer are ductal-, and lobular carcinoma in situ, whose early stages show similar rates of aneuploidy similar to their invasive later stages. This suggests that aneuploidy is a central mechanism in early cancer development and potentially initiation. To test how aneuploidy confers the ability to initiate and drive cancer, we use MCF10A cells in a 3D cell culture model, which reflects many of the physiological properties of human breast lobules. We introduce aneuploidy by downregulating a variety of tumor driver genes that are correlated to CIN in cancer and measure the effects on morphogenesis, expression, tumorigenicity, and chromatin conformation. Using a high-content screening approach, we could identify two genes namely ESPL1 and TOP2A that upon knockdown induce abnormal mitosis resulting in aneuploidy. These cells develop into disorganized acini, which share features of lobular neoplasia, e.g. no hollow lumen and disorganized cell as well as acinus structure. Microarray expression profiles revealed a deregulation of breast cancer related genes including CD24, CD44, ALDH1A3 and CCND1. Interestingly, the chromosome conformation capture (4C-seq) experiments displayed a modified enhancer and CTCF binding landscape for CCND1, which correlated with the upregulation of CCND1. However, the deregulated expression was not sufficient to transform MCF10A cells to form persistent tumors in mice. In conclusion, random aneuploidy influences the chromatin conformation and is capable of deregulating cancer driver genes. This highlights a new mechanism of cancer initiation and development; aneuploidy not only provides a tool for evolution but functions also as a tumor driver.

Item Type: Dissertation
Supervisor: Eils, Prof. Dr. Roland
Date of thesis defense: 5 December 2017
Date Deposited: 19 Jan 2018 08:27
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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