The role of multidrug resistance Protein 1 in the biology of glioblastoma

Author: Mohammed Alnakhli

Alnakhli, Mohammed, 2020 The role of multidrug resistance Protein 1 in the biology of glioblastoma, Flinders University, College of Medicine and Public Health

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Glioblastoma multiforme (GBM) is an aggressive, malignant and incurable brain tumour. The median survival after diagnosis is 15 months. The poor prognosis of GBM tumours is due to multiple factors including intrinsic tumour cell chemo-resistance. The expression of ABC transporter multidrug resistance (MDR) proteins including MRP1 on tumour cells and the endothelium of the c critically contributes to drug resistance of this tumour. Therefore, MRP1 is a potential target for molecular therapy of GBM tumours which, if successfully targeted may impact drug resistance of the tumour and therapeutic outcome for the patient. Other signalling pathways such as the Sonic Hedgehog (SHH) pathway are critical for GBM development however their link to the MDR phenotype is incompletely understood.

In this thesis, it was hypothesised that MRP1 expression is a critical determinant of the malignant and drug resistance phenotype of glioblastoma tumour cells, in association with other key cellular pathways including the Sonic Hedgehog signalling pathway. It was further hypothesised that RNA interference technology could be used to target and knock down MRP1 gene expression in glioblastoma cell lines to assess the contribution of this drug transporter to the biological behaviour of the tumour cells. Specific siRNAs were synthesised and delivered with lipofectamine in vitro to the T98G GBM cell line and demonstrated to be effective in downregulating MRP1 mRNA and protein. A nanoparticle delivery system was then developed and investigated in tissue culture. Optimisation of this delivery system led to the use of PEI (polyethyleneimine) coated nanoparticles preloaded with MRP1 targeting siRNAs that proved effective in a slow sustained release of the siRNA. Down regulation of MRP1 in the T98G GBM cell line was achieved using this delivery system and was found to be associated with reduction in proliferation rate of GBM cells associated with cellular G2 arrest and morphological changes including the accumulation of lipid droplets within the GBM cells. No sustained apoptosis was evident following this treatment. The nanoparticle delivery system was applied in a proof of principle in vivo experiment which demonstrated effective downregulation of MRP1 in xenografted tumour tissues of U87MG cells (82% at 48 hrs and 65% at 72 hrs) and also downregulation of MRP1 in non-tumour organ systems (duodenum and kidney). However, there was no evidence of end organ damage associated with MRP1 downregulation in the examined tissues. To further investigate the observed G2 arrest, a functional inhibitor of MRP1 (MK571) was investigated and found to cause identical results in T98G cells. The expression of SHH pathway members were simultaneously explored using a qRT-PCR approach and it was discovered that key members of this pathway (Gli1, Gli2, Gli3, PATCH 1 and 2, and SUFU) were reduced in their expression following both siRNA inhibition of MRP1 expression and functional inhibition. To further investigate these findings, global expression of mRNA of the siRNA treated cells was explored using an RNAseq approach. This approach failed to corroborate the qRT-PCR data and further experiments will need to be re-performed to assist interpretation of this final piece of data. This thesis has demonstrated that downregulation of MRP1 can be achieved in T98G and U87MG glioblastoma cell lines through RNA interference delivered in a sustained release nanoparticle system and that this is associated with G2 arrest and alteration of the SHH signalling pathway, which is not previously demonstrated.

Keywords: Glioblastoma multiform, MRP1, Sonic Hedgehog, Nanoparticle, Gli1, Gli2, Gli3, PATCH 1 and 2, and SUFU

Subject: Medical Science thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Medicine and Public Health
Supervisor: Bryone Kuss