neuronal conversion of human glioblastoma cells by single neuronal transcription factor

Open Access
- Author:
- Wang, Xin
- Graduate Program:
- Molecular, Cellular and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 03, 2020
- Committee Members:
- Yingwei Mao, Dissertation Advisor/Co-Advisor
Yingwei Mao, Committee Chair/Co-Chair
Timothy J Jegla, Committee Member
Xiaojun Lance Lian, Committee Member
Adam Bleier Glick, Outside Member
Gong Chen, Committee Chair/Co-Chair
Gong Chen, Dissertation Advisor/Co-Advisor
Melissa Rolls, Program Head/Chair - Keywords:
- neuronal conversion
Neurog2
NeuroD1
Ascl1
glioblastoma - Abstract:
- Glioblastoma multiforme (GBM) is the most prevalent and aggressive adult primary cancer in the central nervous system (CNS). Current standard treatment for glioblastoma includes surgical resection and adjuvant radio- or chemotherapy. Other alternative therapeutic approaches are also under intense investigation, such as the emerging immunotherapy. Studies of glioblastoma biology using experimental models, next generation sequencing and other techniques not only enhanced our understanding but also revealed the complexity of this disease. However, with all these advances, marginal progress has been made in glioblastoma treatment over the recent years mainly due to its heterogeneity and highly invasive nature, asking for new interventions in this malicious disease. Considering that neuronal transcription factors are instrumental in nervous system development and in particular neuronal differentiation accompanied by the cell cycle exit, here I propose a potential therapeutic strategy through reprogramming malignant glioblastoma cells to non-proliferative mature neurons using neuronal transcription factors. With an efficient retroviral transduction system, neuronal conversion of U251 human glioblastoma cells was achieved by the overexpression of single neuronal transcription factor Neurogenic differentiation factor 1 (NeuroD1), Neurogenin-2 (Neurog2) or Achaete-scute homolog 1 (Ascl1). Additionally, it was found that majority of the Neurog2- and NeuroD1-converted neurons were glutamatergic, while Ascl1 favored GABAergic neuron generation, suggesting that transcription factors could affect the neuron subtype determination. Functionality of the converted neurons was further confirmed by neuron-specific electrophysiological activities. More importantly, consistent with our hypothesis, the neuronal reprogramming induced by Neurog2 and NeuroD1 led to significant cell proliferation arrest in cultured glioblastoma cells, indicating the dual effects of neuronal transcription factors in neuronal fate decision and proliferation inhibition. To trace the transcriptomic changes and understand the underlying molecular mechanisms, we performed RNA-sequencing (RNA-seq) on human glioblastoma cells with neuronal transcription factor overexpression. It was found that Neurog2 and Ascl1 overexpression triggered distinct transcriptomic responses in U251 human glioblastoma cells. Neurog2 activated a rapid and specific neuronal differentiation program via upregulating the genes encoding for neuron-specific proteins, many of which were neuronal transcription factors. In contrast, Ascl1 drove a broad differentiation program and mainly targeted cell adhesion molecules. Activation of different signaling pathways was also observed upon Neruog2 and Ascl1 overexpression in human glioblastoma cells, suggesting divergent roles of the neuronal transcription factors in the context of glioblastoma. I next examined the feasibility of in vivo neuronal conversion through a xenograft mouse model. U251 human glioblastoma cells and Neurog2 retroviruses were intracranially transplanted in the striatum of Rag1-/- immunodeficient mice. Consistent with the results in cultured cells, efficient neuronal conversion of the transplanted glioblastoma cells was also observed with Neurog2 overexpression. In addition, the in vivo neuronal conversion of glioblastoma cells led to significant reduction of tumor cell proliferation and astrogliosis, reflecting the potential benefits when applying this technology in vivo. In summary, this study demonstrated that overexpression of neuronal transcription factor Neurog2, NeuroD1, or Ascl1 was capable to reprogram human glioblastoma cells to functional neurons. The proliferation arrest and other benefits during neuronal reprogramming in vitro and in vivo suggested that neuronal transdifferentiation might serve as an innovative strategy to impede brain tumor progression.