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Title: Epithelial stromal interaction in breast cancer
Author: Verghese, Eldo Thomas
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2013
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In recent years there has been an increasing awareness of the role played by the microenvironment surrounding breast cancer epithelium in modulating the behaviour of breast tumours. A major component of this microenvironment in breast cancer is the fibroblasts. These are commonly referred to as carcinomaassociated fibroblasts (CAFs). CAFs are phenotypically different from their normal counterpart in normal breast tissue. MicroRNAs, a family of small noncoding RNAs that are key players in the posttranscriptional regulation of mRNAs, have been shown to play a role in controlling the behaviour of cancers. They have been extensively studied in breast cancer epithelial cells and have important roles in breast cancer initiation and progression. My hypotheses were that miRNAs also play roles in controlling the behaviour of breast CAFs and in turn, impact on the behaviour of malignant breast epithelial cells. I used a single patient sample and a co-culture model to examine the expression of miRNAs in breast CAFs and normal fibroblast (NFs). Six miRNAs were consistently down-regulated in CAFs as compared to NFs with a fold change > 10 in both the tissue co-culture model and in patient samples. Of these, miR-26b was significantly down-regulated in CAFs in a further 14 cases of microdissected matched NFs and CAFs from clinical FFPE (Formalin Fixed Paraffin Embedded) samples and consistently in a further 4 cases of matched primary NFs and CAFs in vitro. Functional influences on fibroblasts were examined by overexpressing and ‘knocking down’ miR-26b. Although overexpression of miR-26 in immortalised breast fibroblasts was associated with decreased proliferation and increased apoptosis, this was felt to be not physiologically relevant since the degree of over-expression exceeded the differential expression seen in clinical samples. Reducing the level of miR-26b in immortalised breast fibroblasts resulted in a small decrease in proliferation, and a very notable increase in migration and invasion. Next, the functional influences of fibroblasts with reduced miR-26b on breast cancer epithelial cells were examined. There was no difference in the growth of cancer epithelial cells when they were co-cultured with fibroblasts with low miR-26b as compared to controls. However, these fibroblasts increased the migration and invasion of breast cancer epithelial cells. To identify the potential targets and pathways targeted by miR-26b, I used a combination of mass spectrometry and in-silico analysis. I identified multiple proteins that were differentially expressed between miR-26b knockdown and control fibroblasts. COL12-A1, TNKS1BP1 and CPSF7 were identified as potential targets of miR-26b. I used publically available databases to examine the clinical relevance of these targets in the stroma of breast cancers. Higher expression of these targets was associated with increased rates of recurrence of breast cancer. In addition, pathway analyses of the differentially expressed proteins were enriched for components of the “glycolytic” pathway and “cytoskeleton regulation by RHO GTPase” pathway. In conclusion, I have shown that down regulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and I have identified molecular changes that are downstream of miR-26b.
Supervisor: Hughes, Thomas A. ; Hanby, Andrew M. ; Hull, Mark A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available