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Reprogramming of human cancer cells towards induced pluripotency as a tool to study the influence of epigenetic modifications on the tumor phenotype

Bernhardt, Mathias

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Abstract

Melanoma is the deadliest form of skin cancer. Traditional therapeutic options include surgery, radiation, chemotherapy and immunotherapeutic options. Typically, around 20% of patients develop metastases as the disease progresses, which reduces therapeutic options to provide only palliative benefit in the majority of cases. The genomes of a large number of primary and secondary melanomas were recently sequenced, leading to the identification of melanoma driver mutations. Targeting pathways, activated by cancer-specific genetic alterations, enabled researchers to develop novel therapeutic drugs. Although these drugs effectively fight melanoma cells, cancer cells develop various resistance mechanisms regulated by epigenetic changes, which leads to cancer recurrence in most patients. Nuclear factor-based reprogramming was implemented in melanoma cells to test whether malignant cancer cells can reacquire developmental pluripotency, and moreover to analyze reprogramming-associated epigenetic changes on the tumor cell phenotype. The results showed that the constitutive overexpression of Oct4, Sox2, and Klf4 reprograms melanoma cells into a murine embryonic stem cell-like state. In contrast to fibroblasts, melanoma cells do not require exogenous c-Myc for the induction of a pluripotent stem cell state, characterized by the reactivation of endogenous pluripotency markers and loss of the transcriptional profile of melanoma cells. However, continuous transgene expression is required to maintain an undifferentiated state. When injected into immunocompromised mice, melanoma-derived reprogrammed cells formed teratoma-like tumors containing cell types of all three germ layers, and despite their oncogenic mutations, rarely contained melanoma-like structures. In vitro directed differentiation into neuronal-like and fibroblast-like cells demonstrated that reprogrammed tumor cells acquired the potential to execute terminal differentiation pathways. Although most melanoma cell lines are highly depended on MAPK signaling, reprogrammed tumor cells and their differentiated daughter cells became resistant against BRAF- or MEK-targeting inhibitors, suggesting that epigenetic remodeling processes facilitated therapy resistance against targeted melanoma therapy. Furthermore, global gene expression profiling demonstrated that nuclear reprogramming and subsequent differentiation induced deregulation of tumor suppressors and oncogenes. In conclusion, reprogramming cancer cells allows the investigation of a cancer genome in the context of a specific epigenetic cell state and might help study how alterations in the epigenetic signature control the biological behavior of tumor cells and their response to therapy.

Item Type: Dissertation
Supervisor: Utikal, Prof. Dr. Jochen
Date of thesis defense: 10 March 2015
Date Deposited: 07 Apr 2015 13:14
Date: 2015
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
570 Life sciences
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