Development of therapeutic strategies to identify novel drug targets of DLC1-deleted breast cancers
| dc.contributor.author | Wilson, Nicole A | |
| dc.contributor.examiningcommittee | Murphy, Leigh (Biochemistry and Medical Genetics) | en_US |
| dc.contributor.examiningcommittee | Raouf, Afshin (Immunology) | en_US |
| dc.contributor.supervisor | Mowat, Michael (Biochemistry and Medical Genetics), McManus, Kirk (Biochemistry and Medical Genetics) | en_US |
| dc.date.accessioned | 2021-04-06T20:54:06Z | |
| dc.date.available | 2021-04-06T20:54:06Z | |
| dc.date.copyright | 2021-03-23 | |
| dc.date.issued | 2021-03 | en_US |
| dc.date.submitted | 2021-03-23T21:49:48Z | en_US |
| dc.degree.discipline | Biochemistry and Medical Genetics | en_US |
| dc.degree.level | Master of Science (M.Sc.) | en_US |
| dc.description.abstract | Metastatic disease is the major cause of morbidity and mortality in women with breast cancer. DLC1 codes for a Rho-GTPase activating protein that has both tumour and metastasis suppressor functions. Diminished DLC1 expression occurs in ~50% of breast cancers, thereby making it an ideal candidate for therapeutic interventions that could conceivably target both primary and metastatic disease. In this thesis, I employed a CRISPR-Cas9 gene editing approach to generate syngeneic heterozygous and homozygous DLC1-deleted breast cell lines. Using these cell lines and non-target controls, synthetic lethal (SL) assays were performed to identify candidate genes that could be used as drug targets to selectively kill the DLC1-deleted cell lines. To generate DLC1-deleted breast cell models, I employed CRISPR-Cas9 gene editing in malignant MCF7 and immortalized 184-hTERT breast cell lines. In a population of edited MCF7 cells, I achieved 40-60% reduction in DLC1 protein expression as determined by Western blot analysis. In 184-hTERT cells, clonal heterozygous (DLC1-HET) and homozygous DLC1 deleted (DLC1-KO) cell lines were generated and confirmed via DNA sequencing and Western blot analyses. Candidate drug targets were assessed in the 184-hTERT clonal cell lines for SL interactions with DLC1 deletion. I directly assessed MYH9, DNMT1 and the four members of the FBXW7-SCF complex, using siRNA-based SL assays. MYH9 inhibition resulted in a small but statistically significant decrease in cell numbers between the non-target (NT) control cell line and DLC1-HET. Blebbistatin, a small-molecule inhibitor of the MYH9-containing complex non-muscle myosin II, was assessed as a potential therapeutic agent. Dose-response curves were generated, and the EC50 was compared between cell lines. Blebbistatin treatment resulted in a small decrease in EC50 values in the DLC1-HET compared to NT. In this thesis, DLC1 deleted cellular models were generated and validated. Six preliminary candidate drug targets were assessed in the generated cell lines through SL assays. Further assessment of candidate therapeutic targets using these DLC1-deleted cellular models may result in the identification of novel therapeutic targets of DLC1-deficient tumours that may minimize the morbidity and mortality associated with primary and metastatic disease in breast cancer patients. | en_US |
| dc.description.note | May 2021 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/35406 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.subject | Biochemistry | en_US |
| dc.subject | Genetics | en_US |
| dc.subject | Cancer | en_US |
| dc.subject | Breast Cancer | en_US |
| dc.subject | CRISPR | en_US |
| dc.subject | CRISPR-Cas9 | en_US |
| dc.subject | DLC1 | en_US |
| dc.subject | RhoGAP | en_US |
| dc.subject | Metastasis | en_US |
| dc.subject | MCF7 | en_US |
| dc.subject | Medical Genetics | en_US |
| dc.subject | Gene Editing | en_US |
| dc.subject | Blebbistatin | en_US |
| dc.subject | MYH9 | en_US |
| dc.subject | RhoA | en_US |
| dc.title | Development of therapeutic strategies to identify novel drug targets of DLC1-deleted breast cancers | en_US |
| dc.type | master thesis | en_US |