Ahmed, Tarek Mohamed Abdel Moneim Mohamed Elsaba
(2011)
Role of CD133 in colorectal cancer.
PhD thesis, University of Nottingham.
Abstract
CD133 is a pentaspan transmembrane glycoprotein of ~120 kDa, which was initially used to identify haematopoietic stem cells and, later on, used for the isolation and study of cancer stem cells in many different types of solid tumour including colorectal cancer. Although CD133 expressing cells are thought to represent cancer stem cells, little is known about the exact role of CD133 and the molecular mechanisms underlying control of CD133 expression. This project sought to investigate these questions in colorectal cancer.
Initially the expression of CD133 was tested by immunohistochemistry in a two tissue microarray (TMA) sets consisting of (a) 449 cases of primary colorectal cancer, and (b) 45 cases of primary and matched liver metastases. High CD133 expression was marginally associated with shorter overall survival (OS) (p=0.05, Log-rank test) but no difference in expression was found between primary tumours and corresponding metastases.
Next, the functional activity of CD133 was evaluated in colorectal cancer (CRC) cell lines by knockdown in cell lines with high CD133 expression. In order to identify appropriate cell lines, the expression of CD133 was tested by quantitative RT -PCR in a series of 29 CRC cell lines and 10 samples of normal mucosa and, in selected cell lines, validated by testing for protein expression by flow cytometry. CD133 mRNA was expressed in 24/29 colorectal cancer cell lines with a heterogenous level of expression. 10 cell lines were chosen on the basis of CD133 mRNA expression level to assess the protein level. CD133 mRNA and protein expression were generally correlated (rs = 0.831, p= 0.003, spearman rank correlation coefficient test) although, interestingly, CD133 mRNA level was higher in normal samples compared with that in cancer cell lines and was significantly higher in cell lines derived from metastatic sites than those derived from site of primary tumour (p=0.009; Mann-whitney test). In addition, it was noted that many cell lines had a stable biphasic phenotype containing CD133+ and CD133- cell populations. This allowed functional analysis of CD133 by sorting the two populations.
HT29 was identified as a high expresser of CD133 (95%) and was used for gene-knockdown studies, SW480 had a biphasic population consisting of 42% CD133+ cells and 58% CD133- cells and each population was isolated by cell sorting before functional analysis. Functional assays included proliferation, migration, colony formation and staurosporine induced apoptosis assays. These showed that CD133 expressing cells had greater cell motility (p= 0.04, and p = 0.03, unpaired t-test, for knocked down cells and sorted populations respectively) , enhanced colony forming abilities (p=0.0001, and p=0.003, unpaired t-test for 2D and 3D colony formation respectively using sorted populations only), and increased resistance to staurosporine induced apoptosis (p=0.01, and p=0.008, unpaired t-test, for knocked down and sorted populations respectively) than CD133 negative counterparts. In addition, sorted monophasic populations reverted to a biphasic state in both CD133+/- populations from SW480. Further studies demonstrated that CD133-induced cell motility was independent of E-cadherin, β-catenin, and suggestive of not being regulated by Cten or Wnt, but further work is warranted to verify these results. In addition, regulation of CD133 was partly dependent on STAT3 signalling and on CD133 promoter methylation. Levels of mRNA of some stem cell related genes such as KLF-4, Musashi-1, OCT4, Nanog, and Lgr5 were higher in CD 133 + compared to CD 133 negative cells (p=0.008, p=0.004, p=0.006, p=0.001, and p=0.11; unpaired t-test, respectively)
In conclusion, in CRC, CD133 was found to be a significant prognostic factor which enhances cell motility and is associated with features of "stemness". It is a target of ST AT3 signalling and partly regulated by promoter methylation. More in depth studies are warranted to discover the downstream and upstream targets of CD133 before translating these preclinical and laboratory investigations into clinical management of colorectal cancer.
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