Molecular interactions of polo-like Kinase 1 (PLK1) in colorectal cancer

Abstract

Colorectal cancer (CRC) is one of the most prevalent cancers in Australia with about a third being rectal cancer. Locally advanced rectal cancer is frequently treated with pre-operative radiotherapy (DXT) in concurrent with chemotherapy to reduce the tumour mass for better surgical access, and therefore improving the treatment outcomes. The response to DXT varies amongst individual patients, raising the need of predictive markers to prevent unnecessary toxic therapy. Polo-like kinase 1 (PLK1) is involved in cell cycling and DNA damage response (DDR) and in view of the latter, it may be a potential marker for patient response to chemoradiotherapy. The relationship between PLK1 expression and radiosensitivity remains controversial, particularly in CRC. In addition, the causes of PLK1 overexpression remain unclear although it is associated with oncogenesis. This thesis aims to study the role of PLK1 in radiosensitivity of CRC, the contribution of mutation and DNA methylation to PLK1 expression and the correlation between PLK1 and p21 expression. CRC cell lines (HCT116 and SW48) that are microsatellite unstable and microsatellite stable (Colo320DM and T84) were chosen for the studies. The CRC cells were treated with PLK1-specific siRNA and ionising radiation (IR) and then subjected to PLK1 expression, cell survival, apoptotic and cell cycling analyses. Data analysis from Catalogue of Somatic Mutations in Cancer (COSMIC) and Sanger sequencing of PLK1 gene were performed in the genetic study. The DNA methylation study involved mass spectrometry analysis and pyrosequencing of DNA from the CRC samples. The correlation between PLK1 and p21 was studied by immunohistochemical (IHC) staining on tissue microarray (TMA) of CRC patient cohort. Depletion of PLK1 additively enhanced the effect of IR on cell survival, apoptosis and cell cycle arrest in HCT116, SW48 and Colo320DM, but did not radiosensitise the cells. COSMIC data showed a low incidence of mutation in CRC, but the mutation c.1010A>G (p.R3370Q) at the motif for ubiquitination and proteasomal degradation of PLK1 may elevate the PLK1 stability. Sanger sequencing detected mutations of PLK1 in HCT116 and SW48 but they are not functionally associated. DNA methylation studies concluded that PLK1 expression is likely to be independent of CpG methylation. IR induced a transient increase of PLK1 expression in all the microsatellite unstable cell lines with HCT116 and SW48 downregulated later. Lastly, IHC of CRC patient cohort TMA demonstrated the weak association between PLK1 and p21 expression.

Date of Award	2017
Original language	English