UVM Theses and Dissertations
Format:
Print
Author:
Long, Patrick M.
Dept./Program:
Neuroscience Graduate Program
Year:
2013
Degree:
Ph. D.
Abstract:
Glioma is the most common malignant primary adult brain cancer and is associated with poor prognosis. The standard oftherapy (i.e. maximal tumor resection followed by radiotherapy and chemotherapy) extends the median survival for high-grade glioma to only 14 month. Limited therapeutic efficacy is thought to be due to the presence of a subset of tumor cells termed glioma stem-like cells (GSCs), which are highly treatment resistant and are the primary drivers ofpost-surgical tumor recurrence. The development of therapies that are efficacious against the GSC population is therefore ofsignificant clinical importance.
Histone acetylation is a widely recognized mechanism of epigenetic regulation. Progressive histone hypoacetylation leads to chromatin compaction and transcriptional silencing oftumor suppressor genes thus facilitating tumor cell transformation. Acetylcoenzyme A (acetyl-coA) is a necessary cofactor for histone acetylation reactions and fluctuations in acetyl-coA biovailability regulate histone acetylation state.
N-acetyl-aspartate (NAA) is among the most abundant sources of acetyl-coA in the brain. NAA is catabolized by the amino acid deacetylase aspartoacylase (ASPA) to generate acetate, which is subsequently converted to acetyl-coA by acetyl-coA synthetase enzymes. NAA may alternatively be converted to the small peptide neurotransmitter Nacetyl-aspartyl-glutamate (NAAG), which represents an additionally form of acetate storage. Studies of ASPA deficient rodents reveal significant reductions in brain acetate levels coincident with increased neural stern and progenitor cell proliferation. Insomuch as oligodendroglioma and astrocytoma tumors show reduced ASPA expression concordant with a loss of NAA and NAAG bioavailability, we hypothesized that glioma tumors possess an acetate deficiency that facilitates tumor cell malignancy by enabling histone hypoacetylation.
This dissertation, therefore, tested the hypothesis that acetate supplementation would exert a tumor suppressive effect on GSCs. This strategy was undertaken using two approaches. First, acetate supplementation in the forms of NAA or NAAG was tested for its efficacy in inducing GSC differentiation and growth arrest. As ASPA is decreased in glioma and thus may not catabolize NAA, an alternative acetate source not dependent on ASPA catalytic activity was tested. This second approach was performed using the FDA approved food additive glycerol triacetate (GrA) as a means for acetate delivery in the treatment of GSCs.
In contrast to our hypothesis, NAA and NAAG increased growth of oligodendroglioma GSCs and attenuated serum-induced differentiation of astrocytoma GSCs. While ASPA expression is reduced in the tumor bulk, ASPA is expressed by GSCs and was enriched in the nuclei of GSCs that displayed a mesenchymal tumor phenotype and were differentiation resistant. ASPA exhibited a regulated pattern of subnuclear localization throughout the cell cycle. Furthermore the expression of novel immunoreactive ASPA species that partitioned to GSC nuclei were identified, suggesting differential processing of ASPA in the nuclear compartment.
Unlike NAA or NAAG, acetate supplementation in the form of GTA exerted a profound cytostatic effect on oligodendroglioma GSC growth. GrA-induced GSC cytostasis was not accompanied by increased cell death or GSC differentiation. Combined treatment of GTA with the chemotherapeutic agent temozolomide in orthotopic xenografts of oligodendroglioma GSCs resulted inreduced tumor volume and increased survival of immounodecicient mice compared to temozolomide treatment alone.
Collectively, these studies reveal oncogenic, rather than tumor suppressive, properties for NAA and NAAG, and argue against the use of these metabolites as a glioma therapeutic. Additionally, these studies provide a foundation for future investigations of ASPA's nuclear localization in contributing to GSC differentiation resistance. Importantly, these studies are the first to identify GTA as a novel therapeutic agent for the treatment of glioma, and future investigations of GTA's therapeutic potential in the clinical setting are warranted.
Histone acetylation is a widely recognized mechanism of epigenetic regulation. Progressive histone hypoacetylation leads to chromatin compaction and transcriptional silencing oftumor suppressor genes thus facilitating tumor cell transformation. Acetylcoenzyme A (acetyl-coA) is a necessary cofactor for histone acetylation reactions and fluctuations in acetyl-coA biovailability regulate histone acetylation state.
N-acetyl-aspartate (NAA) is among the most abundant sources of acetyl-coA in the brain. NAA is catabolized by the amino acid deacetylase aspartoacylase (ASPA) to generate acetate, which is subsequently converted to acetyl-coA by acetyl-coA synthetase enzymes. NAA may alternatively be converted to the small peptide neurotransmitter Nacetyl-aspartyl-glutamate (NAAG), which represents an additionally form of acetate storage. Studies of ASPA deficient rodents reveal significant reductions in brain acetate levels coincident with increased neural stern and progenitor cell proliferation. Insomuch as oligodendroglioma and astrocytoma tumors show reduced ASPA expression concordant with a loss of NAA and NAAG bioavailability, we hypothesized that glioma tumors possess an acetate deficiency that facilitates tumor cell malignancy by enabling histone hypoacetylation.
This dissertation, therefore, tested the hypothesis that acetate supplementation would exert a tumor suppressive effect on GSCs. This strategy was undertaken using two approaches. First, acetate supplementation in the forms of NAA or NAAG was tested for its efficacy in inducing GSC differentiation and growth arrest. As ASPA is decreased in glioma and thus may not catabolize NAA, an alternative acetate source not dependent on ASPA catalytic activity was tested. This second approach was performed using the FDA approved food additive glycerol triacetate (GrA) as a means for acetate delivery in the treatment of GSCs.
In contrast to our hypothesis, NAA and NAAG increased growth of oligodendroglioma GSCs and attenuated serum-induced differentiation of astrocytoma GSCs. While ASPA expression is reduced in the tumor bulk, ASPA is expressed by GSCs and was enriched in the nuclei of GSCs that displayed a mesenchymal tumor phenotype and were differentiation resistant. ASPA exhibited a regulated pattern of subnuclear localization throughout the cell cycle. Furthermore the expression of novel immunoreactive ASPA species that partitioned to GSC nuclei were identified, suggesting differential processing of ASPA in the nuclear compartment.
Unlike NAA or NAAG, acetate supplementation in the form of GTA exerted a profound cytostatic effect on oligodendroglioma GSC growth. GrA-induced GSC cytostasis was not accompanied by increased cell death or GSC differentiation. Combined treatment of GTA with the chemotherapeutic agent temozolomide in orthotopic xenografts of oligodendroglioma GSCs resulted inreduced tumor volume and increased survival of immounodecicient mice compared to temozolomide treatment alone.
Collectively, these studies reveal oncogenic, rather than tumor suppressive, properties for NAA and NAAG, and argue against the use of these metabolites as a glioma therapeutic. Additionally, these studies provide a foundation for future investigations of ASPA's nuclear localization in contributing to GSC differentiation resistance. Importantly, these studies are the first to identify GTA as a novel therapeutic agent for the treatment of glioma, and future investigations of GTA's therapeutic potential in the clinical setting are warranted.