Evaluating the effect of LRP/LR antibodies in colorectal carcinoma mouse models and assessing the molecular mechanism of LRP/LR on apoptotic pathways in colorectal carcinoma cells

Vania, Leila
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Cancer remains one of the leading causes of death around the world with its incidence and mortality rates constantly increasing. Tumourigenic cells are characteristically seen to overexpress the 37kDa/67kDa laminin receptor (LRP/LR) compared to their normal cell counterparts. LRP/LR is involved in promoting tumourigenic processes such as cellular viability maintenance and apoptotic evasion. Thus, the first main aim of this study was to investigate the role of LRP/LR in cellular viability of early (SW-480) and late stage (DLD-1) colorectal carcinoma cells and thereafter assess the molecular mechanism of LRP/LR on apoptotic pathways upon siRNA-mediated down-regulation of LRP/LR in vitro. The data from the study was analysed using a one-way ANOVA, followed by a two-tailed student’s t-test with a confidence interval of 95%. To further validate the data, the Bonferroni post-hoc test was applied, with p-values of less than 0.05 considered to be significant. Results from the in vitro study showed upon siRNA-mediated down-regulation of LRP/LR, the viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells was significantly reduced through increased levels of apoptosis apparent by compromised membrane integrity as determined by Annexin-V/PI assays and confocal microscopy, respectively. Caspase-3 activity assays were performed to confirm apoptosis, where both cell lines exhibited significantly high caspase-3 activity. In addition, down-regulation LRP/LR resulted in a significant increase in caspase-8 and -9 activity in both colorectal carcinoma cell lines. Cell cycle analysis showed that LRP/LR down-regulation resulted in a significant increase in the sub G0/G1 apoptotic phase of DLD-1 cells, when compared to non-transfected cells, confirming the occurrence of apoptosis. To evaluate the mechanistic role of LRP/LR, proteomic analysis of pathways involved in proliferation and apoptosis were further investigated with the MAPK and Apoptotic Proteome Profiler Antibody Arrays™ as well as SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra). Upon siRNA-mediated down-regulation of LRP/LR, various pro-apoptotic proteins including Bax, cleaved caspase-3 and p53 as well as specific proteins in the MAPK cell signalling pathway such as Chk-2 and AMPKα2 were significantly up-regulated in the DLD-1 cells. In addition, several anti-apoptotic proteins were seen to be significantly down-regulated upon siRNA-mediated down-regulation of LRP/LR including xIAP, Claspin and Survivin. The results obtained from the Proteome Profiler Antibody Arrays™ were confirmed through qPCR, by investigating mRNA expression levels of specific proteins. SWATH-MS analysis showed that siRNA-mediated down-regulation of LRP/LR led to significant changes in the proteome of DLD-1 colorectal cancer cells, revealing new roles of the protein as well as confirming its existing roles. Moreover, it showed that LRP/LR may alter components of the MAPK, p53-apoptotic as well as autophagic signalling pathways to aid colorectal cancer cells in continuous growth and survival. Telomerase activity and telomerase-related proteins were also investigated using qPCR and western blotting, respectively. A significant decrease in telomerase activity as well as decreases in phospho-TERT and telomeric repeat-binding factor 2 (TFR-2) protein expression levels were observed in the DLD-1 cells upon down-regulation of LRP/LR. In addition to its role in cell viability and apoptotic evasion, LRP/LR has also been found to be involved in enhancing tumour cell adhesion and invasion, key points of metastasis. However, previous studies revealed that the application of the anti-LRP/LR specific antibody IgG1- iS18 led to a significant reduction in the metastatic potential of colorectal carcinoma cells in vitro. Therefore, the second aim of the present study was to investigate the significance of blocking LRP/LR with the IgG1-iS18 antibody and to determine whether it will be effective in the treatment of colorectal carcinoma in vivo using an MF-1 nude mice model. Results from several in vivo pilot studies performed showed that all mice were able to successfully develop tumours upon intraperitoneal injection of HT-29 colorectal cancer cells. However, it was found that despite various changes in the concentration as well as the route of administration of the IgG1-iS18 antibody, there was no significant effect seen in tumour growth/shrinkage and metastasis. Due to the data obtained from the pilot studies and time constraints, the full study was not performed. Although the mechanism of action of LRP/LR is far from being understood, findings from this study show that LRP/LR is critically implicated in apoptosis and cell viability maintenance. The current study therefore suggests that siRNA-mediated down-regulation of LRP/LR could be a possible therapeutic strategy for the treatment of colorectal cancer
Thesis submitted in fulfilment of the requirements for the degree Philosophiae Doctor in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020