Investigating the role of the apelinergic system in glioblastoma

Elucidating the molecular signalling circuitry that underpins the pathogenesis of cancers is critical to understanding and developing effective treatment paradigms for cancer. The apelinergic system is a signalling pathway primarily consisting of the apelin peptide (APLN) and the apelin receptor (APLNR). The apelinergic system has been implicated in the pathophysiology of several cancers. However, there have been very few reports regarding the role of the apelinergic system in the brain cancer glioblastoma. Glioblastoma is a highly recalcitrant malignancy with a poor prognosis which makes understanding the underlying molecular pathogenesis critical for developing new treatments. The goal of this thesis was to investigate the role of the apelinergic system in glioblastoma. This primary goal was divided into three specific aims that comprised of the following.  i) Determine the expression of APLN and APLNR mRNA in glioblastoma and glioblastoma-derived cell lines as well as use public data to investigate the expression of APLN and APLNR in other astrocytic and oligodendroglial tumours.  ii) Develop a cellular model for the apelinergic system in glioblastoma. iii) Determine the response of the apelinergic system to common glioblastoma stressors.  APLN, but not APLNR, mRNA expression was elevated in glioblastoma compared to normal tissue. Analysis of mRNA expression from several public data sources through the GLIOVIS portal demonstrated that both APLN and APLNR were upregulated in glioblastoma relative to lower grade tumours. Significantly, it was also noted that APLN expression was strongly associated with regions of hypoxia whilst APLNR expression was elevated in astrocytes, tumour cells and vascular cells.  The mRNA expression of APLNR and APLN was high in resected glioblastoma tissue but 100 – 1000 fold lower in glioblastoma-derived cell lines. The low expression of APLNR in glioblastoma-derived cells suggested that these cell lines would not be a suitable model for testing the apelinergic system in glioblastoma. To overcome this limitation, the established glioblastoma-derived cell line U87MG was stably transfected with a plasmid expressing an APLNR-GFP fusion protein to recapitulate the expression of APLNR seen in resected tissue, and this cell line was used as a model of the apelinergic system in glioblastoma. This U87.APLNR cell line demonstrated that exogenous [Pyr1]apelin-13 could induce migration in the transwell migration assays, indicating a possible role in glioblastoma tumour cell migration. Glioblastomas like other solid tumours are exposed to significant stresses from sources such as hypoxia, nutrient deprivation and chemotherapeutic treatment. The apelinergic system has been reported to protect against cell death caused by several stressors, including hypoxia and glucose deprivation, but this has not been investigated in glioblastoma cells. The effects of hypoxia and glucose deprivation and chemotherapeutic treatments were investigated in regard to mRNA expression of APLN and APLNR in glioblastoma-derived cell lines. APLN mRNA expression was upregulated in hypoxic (1% oxygen) conditions. However, the addition of exogenous [Pyr1]apelin-13 did not affect U87.APLNR cell number when cultured under hypoxic, glucose deprived or combined oxygen-glucose deprived conditions. This may suggest hypoxia-mediated APLN expression has a role outside of protection. No significant changes in mRNA expression of APLN or APLNR were detected in response to the chemotherapeutic agents doxorubicin and temozolomide. Treatment of U87.APLNR cells with exogenous [Pyr1]apelin-13 failed to protect against chemotherapeutic-induced cell death. It was concluded that the treatment of U87.APLNR cells with [Pyr1]apelin-13 did not protect against hypoxic, hypoglycaemic, combination or chemotherapeutic induced stress. Collectively, this thesis presents an exploration of APLN and APLNR expression in glioblastoma. It provides the first evidence of hypoxia-induced APLN expression in glioblastoma-derived cell lines. The results of this research suggest, however, that it is unlikely that the apelinergic system has a significant role in protecting against cell stress in glioblastoma but suggests that hypoxia-regulated APLN may play a critical role in glioblastoma, possibly as a signalling molecule, and this warrants further research.