Distribution and role of TET-mediated DNA and RNA hydroxymethylation in breast cancer and normal breast epithelium
File(s)
Author(s)
Wen, Jinrui
Type
Thesis
Abstract
Breast cancer (BC) is the most frequently diagnosed malignancy in women. Epigenetic modifications, involving DNA, RNA, and histone modulation, result in the alteration of gene expression. Among these epigenetic modifications, 5-hydroxymethylcytosine (5hmC) has emerged as being relevant in various biological and pathological processes. In particular, there is evidence that 5hmC is involved in cancer progression and may possibly function as a prognostic biomarker.
The first aim of this study was to validate 5hmC distribution and hypothesise its modulatory role in the transcription and translation of DNA and RNA in BC via immunohistochemistry (IHC) and immunocytochemistry (ICC). Through IHC and ICC, I detected cytoplasmic 5hmC for the first time in BC. RNA hydroxymethylation was detected by dot blot analysis of the human mammary cell lines MCF10A and BT474, a finding previously reported in other cells, such as colorectal cancer, but not in breast cancer. A stratification of the samples in different cancer subtypes showed that the proportion of positive cells of cytoplasmic 5hmC was significantly higher only in ER-negative cancer epithelium compared to the normal matched epithelium.
In further investigations, I examined whether 5hmC levels were associated with a specific TET protein in BC. Here, IHC experiments were performed on the same samples. The distribution of cytoplasmic TET2 showed correlations with the distribution of 5hmC and this may have prognostic significance. Following the knockdown of TET mRNA expression by siRNA (in BT474), down-regulation of the TET2 transcript led to lower cell proliferation while loss of TET1 or TET3 expression was associated with increased proliferation.
In conclusion, I suggest that 5hmC may have a role in the development of certain sub-types of BC and TET2 is of particular relevance here.
The first aim of this study was to validate 5hmC distribution and hypothesise its modulatory role in the transcription and translation of DNA and RNA in BC via immunohistochemistry (IHC) and immunocytochemistry (ICC). Through IHC and ICC, I detected cytoplasmic 5hmC for the first time in BC. RNA hydroxymethylation was detected by dot blot analysis of the human mammary cell lines MCF10A and BT474, a finding previously reported in other cells, such as colorectal cancer, but not in breast cancer. A stratification of the samples in different cancer subtypes showed that the proportion of positive cells of cytoplasmic 5hmC was significantly higher only in ER-negative cancer epithelium compared to the normal matched epithelium.
In further investigations, I examined whether 5hmC levels were associated with a specific TET protein in BC. Here, IHC experiments were performed on the same samples. The distribution of cytoplasmic TET2 showed correlations with the distribution of 5hmC and this may have prognostic significance. Following the knockdown of TET mRNA expression by siRNA (in BT474), down-regulation of the TET2 transcript led to lower cell proliferation while loss of TET1 or TET3 expression was associated with increased proliferation.
In conclusion, I suggest that 5hmC may have a role in the development of certain sub-types of BC and TET2 is of particular relevance here.
Version
Open Access
Date Issued
2022-11-05
Date Awarded
01/11/2023
License URL
Advisor
Stebbing, Justin
Cereser, Biancastella
Sponsor
Action Against Cancer
Publisher Department
Department of Surgery & Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)