Identification of Therapeutic Targets and Molecular Markers for Glioblastoma Stem Cells.
Kulkarni, Shreya.
2016
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Abstract: Glioblastoma Multiforme (GBM) is the most common type of malignant brain cancer with a median survival of only 14 months post diagnosis. The current standard of care for all the patients is maximal surgical resection followed by radiation and chemotherapy with temozolomide, a DNA alkylating agent. This treatment reduces the tumor bulk, but is not curative as recurrence is very common. Th... read moree GBM tumor cell population is heterogeneous and a small percentage of the tumor cell population, known as GBM stem cells (GBM-SCs), has the capacity to initiate and sustain tumor growth as well as the ability to survive in hypoxic tumor niches. Hypoxia drives clonogenicity of GBM-SCs and increases their ability to resist chemo and radiotherapy. GBM-SCs can be isolated using cell surface markers such as CD133. However, these markers are not ideal, as they do not detect all the cancer stem cell populations and their expression can change in response to the external microenvironment, which may lead to confounding results. Due to the high mortality associated with these tumors, there is an urgent need for the development of new therapeutics against GBM and especially ones that specifically target the GBM-SC population. Further, the lack of good GBM stem cell markers is a hindrance to our ability to identify and study these cells. In this thesis, I have identified novel therapeutic targets for GBM-SCs and have characterized novel molecular markers for GBM-SCs. First, I have used a genome-wide RNAi approach (~10,000 genes) to screen and identify genes important for cellular growth and survival, which could potentially be used to target the GBM stem cell population. Using this unbiased RNAi screening approach, I identified several genes important in GBM stem cell growth and survival in hypoxic (1% oxygen) and normoxic (21% oxygen) conditions. I identified 81 essential genes, which are required for GBM-SC growth under both normoxic and hypoxic conditions in two different GBM-SC cell lines. Interestingly, only about a third of the essential genes were common to both cell lines. This indicates a high degree of variability between the cell lines and suggests that for a given tumor the majority of potential targets may be tumor specific highlighting the need for personalized therapies. 30% of the hits under hypoxic conditions were specific to this oxygen environment suggesting that some genetic targets are dependent on the oxygen environment. In addition to revealing genetic hits that are essential genes already implicated in GBM such as CDK4, KIF11 and Ran, the screen also identified genes that have not been previously implicated in GBM stem cell biology. One such gene, the Serum and Glucocorticoid regulated Kinase -1 (SGK1) was selected for further investigation. SGK1 scored as a hit in two different GBM-SC lines in the pooled RNAi screen. I have validated SGK1 as a gene important in GBM-SC survival using shRNA mediated knockdown, CRISPR/Cas9 gene modification, as well as pharmacological inhibition of the kinase. I show that SGK1 depletion induces cell death specifically in the GBM-SCs and not in traditional serum-dependent glioma cell lines. This effect is also exclusive to the undifferentiated state of the GBM-SCs. Finally, to address the need for identification of better molecular markers for GBM-SCs, I characterized a panel of GBM-SC specific monoclonal antibodies that our laboratory had generated previously in a collaborative effort with Douglas Jefferson's laboratory and Cell Essentials. The antigen for these antibodies is lost upon differentiation of GBM-SCs and a similar pattern was observed for staining of undifferentiated and differentiated embryonic stem cells for mAb 7-18. We have identified the antigen for mAb 7-18 by immunoaffinity purification and mass spectrometry to be the Coxsackie virus and Adenovirus Receptor (CXADR), which is currently being investigated for its utility as a stem cell marker for GBMs. All these antibodies have also been evaluated for their possible use as toxin conjugated mAb therapeutics. We have shown that these antibodies bind cell surface antigens, are internalized, and can detect their antigen in human GBM tissue samples. Thus, these antibodies may be novel drug development candidates for identifying and possibly targeting GBM-SC in human GBMs. The results of this thesis have advanced our knowledge of GBM biology by identifying new genetic targets, which may be therapeutically relevant and by characterizing new molecular markers to further our understanding of GBM stem cells.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisor: Brent Cochran.
Committee: Gary Sahagian, Michael Forgac, and Charles Stiles.
Keyword: Molecular biology.read less - ID:
- 794088700
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