Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.786383
Title: Investigation of CNR1 as a tumour suppressor gene at 6q15 in prostate cancer
Author: Mohammad, Eiman
ISNI:       0000 0004 7971 8479
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2019
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Abstract:
Molecular genetic analysis by our team and others revealed a frequent deletion of the 6q15 region in prostate cancer (PCa), suggesting the presence of one or more tumour suppressor gene(s) (TSG) within this region, whose inactivation may play a role in prostate carcinogenesis. Previously we have shown that the cannabinoid receptor 1 (CNR1) gene, located at 6q15, was down-regulated in PCa cell lines and one clinical PCa sample at the mRNA level compared to normal tissue, suggesting CNR1 as a potential TSG in PCa. I, therefore, aimed to investigate whether CNR1 may act as a TSG in prostate cancer. I sequenced the coding and 1 kbp promoter regions of this gene in five prostate cancer cell lines, 22RV1, LNCaP, DU145, PC3 and VCaP. No mutations were found, which is consistent with these cells having a potentially functional CNR1. A 6bp polymorphism sequence was found on the promoter region, with the shorter allele more frequently found in cancer samples. The 6 bp polymorphism causes the addition of another transcription factor binding sites for each transcription factors (TFII-1, STAT4, c-Ets-1, Elk-1), but no association of the polymorphism with prostate cancer risk was found. Further investigation will determine its impact on the CNR1 gene translation and functionality in PCa. Mutation analysis was further performed using combined fluidigm amplification and next generation sequencing on 73 PCa clinical samples. No mutations in CNR1 coding and promoter regions were identified. CNR1 expression at mRNA levels was investigated in prostate cancer cell lines, 22RV1, LNCaP, DU145, PC3 and VCaP cells. Using real-time PCR analysis, I found CNR1 was highly expressed at mRNA level in 22RV1, LNCaP, DU145, while lower levels of expression were detected in PC3 and VCaP cells compared to PNT1a immortalised prostate epithelial cells. The protein expression of CNR1 receptors was investigated in PCa cell lines using different commercial anti-CNR1 antibodies. The specificity of the CNR1 antibody (Ab23703) was confirmed using two approaches including Knocked-down of CNR1 protein in prostate cancer cell line 22RV1 and inhibition of CNR1 glycosylation protein in the prostate cancer cell line LNCaP. These data confirmed that ab23703 antibody proved to be specific for CNR1 receptors and can be used for further functional studies to investigate the role of CNR1 in prostate. Using ab23703 antibody, the expression of CNR1 at the mRNA level generally correlated with the protein level in the prostate cancer cell line. To further investigate the role of CNR1 as a TSG in prostate cancer cells, I employed various functional assays including cell viability (MTS), migration (Transwell), scratch-wound and colony formation assays. These studies showed that CNR1 knockdown in DU145 cell lines caused a significant increase in cell viability, cell migration and invasion. Furthermore, cell cycle analysis showed that CNR1 knockdown caused a decrease in G0/1 phases and an increase in G2/M phases of DU145 cells. These results suggest that CNR1 may be involved in the suppression of prostate cancer cell growth and invasion. Next, I investigated whether targeting CNR1 with cannabinoid agonists would have a potential for treating prostate cancer. Addition of the CNR1 agonist (HU210) to the prostate cancer cells 22RV1, LNCaP, DU145, and PC3 resulted in a significant decrease in cell viability in a dose-dependent manner as determined by MTS assays. The reduction in cell viability induced by the cannabinoid HU210 was significant (**P < 0.01) and was prevented by the CNR1 antagonist (R)-SLV-319 in all evaluated cell lines, demonstrating that HU210-induced cell killing is initiated through activation of the cannabinoid receptor and that CNR1 mediates anti-proliferative effects in response to agonists. I further demonstrated that HU210 reduces the viability of the (22RV1, LNCaP, DU145, PC3) cells through induction of apoptosis by Western blot analysis of caspase-3 and PARP proteins. My findings demonstrate that CNR1 may play an important role in proliferation, migration and invasion of prostate cancer cells. These results suggest that CNR1 may act as a tumour suppressor gene in prostate cancer cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.786383  DOI: Not available
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