ROR1 and Rab27b – novel biomarkers for chemoresistance in ovarian cancer

Ovarian cancer is one of the most lethal cancers of the female reproductive system. Most patients relapse with chemo-resistant form of the disease after chemotherapy. Preliminary research profiled drug resistant (IGROVCDDP) and sensitive (KB-5-5-11) cell line models using whole genome Affymetrix expression arrays which identified novel biomarkers; ROR1 and Rab27b. These were validated in publicly available microarray datasets and through PCR-based pilot study on ovarian cancer tissue blocks. The aim of this project was to investigate ROR1 and Rab27b as biomarkers for chemoresistance in ovarian cancer. Ovarian cancer cell line panel was assembled comprising of; HEY, SKOV-3, OVCAR-3 and OAW42. The IC50 doses of four drugs; cisplatin, carboplatin, taxol and talazoparib for the cell lines were determined. qPCR was carried out to investigate gene expression levels of ROR1, Rab27b and EMT markers in response to drug treatments for each cell line. ELISA’s and Immunocytochemistry were carried out to investigate protein expression and localization of the ROR1and Rab27b in response to drug treatments. An invasion assay was carried out to establish an invasion profile of the cell line panel. Knockdown of ROR1(and ROR2) in the resistant and sensitive cell lines was carried out to study the effects on chemo-resistance. Clinical tissue samples obtained from Westmead Hospital, Sydney, Australia were also stained and scored for ROR1(and vimentin). Kaplan-Meir survival analysis was carried out to investigate the impact of ROR1 (and vimentin) on patient outcomes. HEY cells were found to be the most resistant (p<0.05) and invasive (p<0.001) while OVCAR-3 was the opposite (p<0.001). Gene expression assays revealed ROR1 was highest in the resistant cell line. The protein assays revealed ROR1 expression was correlated with chemoresistance (R2 = 0.99) and invasion (R2 = 0.82). Knockdown studies in HEY and OVCAR-3 cells revealed a significant re-sensitisation to platinum-based drugs when undergoing simultaneous ROR1 and ROR2 silencing (p<0.05). Tissues stained for ROR1 and Vimentin showed a poor overall survival in ovarian cancer patients with high ROR1 and vimentin scores. Rab27b gene and protein assays revealed varying expression patterns with drug treatment indicating the need for further studies to better understand its role in chemoresistance. Overall, ROR1 is a promising predictive biomarker for chemoresistance which will be invaluable in the field of personalized medicine for ovarian cancer.

Level dc:type.qualificationlevel

PhD thesis

Grantor dc:publisher.institution

Middlesex University

Year dc:date.issued

2020

Author dc:creator

• Syed Shafat, E.

Identifier

oai:repository.mdx.ac.uk:8q1zz

OAI identifier oai:identifier

oai:repository.mdx.ac.uk:8q1zz