Role of Transcription Factors AP-2 and NFI in Development and Glioblastoma

• Author / Creator

  Saket Jain

• Gene regulation pathways involved in embryonic development are commonly implicated in cancer. Transcription factors play key roles in all aspects of development including cell proliferation, migration and differentiation. Aberrant expression of many developmentally-regulated transcription factors contributes to many malignancies. In this thesis, we studied the role of transcription factor family AP-2 in retinal development and glioblastoma (GBM) and the role of transcription factor family NFI in GBM. Four of the five members of the AP-2 family (AP-2α, AP-2β, AP-2 and AP-2) have previously been shown to be expressed in developing retina. In Chapter 2, we show that the fifth member of the AP-2 family, AP-2ε, is also expressed in the developing mammalian retina. Our data point to a specialized role for AP-2 in a subset of amacrine cells, with AP-2 being restricted to the GABAergic amacrine lineage. AP-2 is co-expressed with AP-2, AP-2β and AP-2 in subsets of amacrine cells, suggesting roles for both AP-2 homodimers and AP-2 heterodimers in the regulation or AP-2 target genes in the retina. Our work suggests spatially- and temporally-coordinated roles for combinations of AP-2 transcription factors in amacrine cells during retinal development.
  Several studies have implicated aberrant regulation of AP-2 with cancer. In Chapter 4, we examined the role of AP-2 in GBM progression. GBMs are the most aggressive brain cancers with a dismal prognosis. Despite aggressive treatment including surgery followed by radiotherapy and chemotherapy the median survival remains ~14 months. In low grade astrocytoma, AP-2α is primarily found in the nucleus, whereas in GBM, it has a cytoplasmic pattern. Based on our results, three members of the AP-2 family, AP-2α, AP-2β, and AP-2, are widely expressed in GBM cell lines and patient-derived neurosphere cultures. Interestingly, AP-2β levels are particularly high in patient-derived neurosphere cultures when compared to adherent cells derived from the same patient. Furthermore, AP-2β primarily localizes to the nucleus of cells cultured under neurosphere conditions compared to cells cultured under adherent conditions. Depletion of AP-2β results in reduced expression of stem cell markers Nestin and SOX2. As well, cell migration is significantly reduced upon AP-2β depletion in patient-derived GBM cultures. As hypoxia is a hallmark of GBM tumours, we examined the effect of AP-2β on cell migration markers and well as stem cell markers under hypoxia. Overall, our results indicate a role for AP-2β in stem cell maintenance in GBM.
  Next, we studied the role of NFI, in GBM. The NFI family consists of four family members NFIA, NFIB, NFIC and NFIX. In the developing CNS, NFI family members are involved in glial cell differentiation. Studies from our lab and other labs suggest a role for NFIs in GBM. In Chapter 3, we used ChIP-on-chip to identify additional NFI targets in GBM cells. Of ~400 putative targets identified using this approach, we focused on HEY1, a Notch effector gene associated with maintenance of neural stem cells. We showed that all four NFIs can bind to the NFI recognition sites in the HEY1 promoter and that NFI negatively regulates the expression of HEY1. We further showed that depletion of HEY1 in GBM cells results in reduced cell proliferation and increased cell migration. We also found a correlation between elevated HEY1 levels and expression of B-FABP, a stem/progenitor cell marker in GBM cells, and also showed that HEY1 depletion results in increased levels of astrocyte differentiation marker GFAP. Overall our results indicate that NFI negatively regulates HEY1 and expression of HEY1 is associated with the expression of stem cell markers in GBM.

• Subjects / Keywords

  • Transcription factors; AP-2; NFI; Amacrine cells; Tumor neurosphere; stem cells; transcription; gel shifts; chromatin immunoprecipitation; HEY1; Glioblastoma

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3VD6PM5S

• License

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