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Title: SIP1/ZEB2-induced epithelial mesenchymal transition promotes metastasis and alters chemokine (C-C motif) ligand 5 expression to modulate the tumour microenvironment in colorectal cancer
Author: Al Saihati, Hajir
ISNI:       0000 0004 6500 6091
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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Epithelial mesenchymal transition (EMT) is a critical trans-differentiation program driving cancer metastasis. Patients showing signs of invasive cancer or the presence of distant metastasis have a poor prognosis. Another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. Thus I hypothesized that the EMT and anti-tumor response could be linked via altered secretion of soluble factors by metastatic cells. Colorectal cancer (CRC) cell lines and SIP1-inducible DLD cells were grown in DMEM. The induction of the SIP1 gene was carried out using doxycycline for 3 days. The EMT status of the CRC cell lines were assessed by preforming western blotting, immunofluorescence and RT-PCR for EMT biomarkers. Cytokine/chemokine expression in SIP1 inducible DLD cells was analyzed using commercially-available antibody arrays. Validation of the selected chemokine (CCL5) was carried out using sandwich ELISA as well as RT-PCR, with the CCL5 expression level analysed in a panel of CRC cell lines using the same techniques. The CCL5 promoter was then cloned into pGL3. The mechanism of action of ZEB1/2 on the CCL5 promoter was studied by luciferace assay and chromatin immunoprecipitation (ChIP). CCL5 coding region was cloned into pcDNA3.1 and stably transfected into DLD-1 cells. DLD-1 cells over-expressing CCL5/RANTES were injected orthotopically into SCID mice, and metastasis was investigated by immunohistochemistry (IHC). The relationship between infiltrating T lymphocytes (TILs) and the expression of both CCL5/RANTES and SIP1 was studied in 75 CRC patients by IHC and tissue micrroarray. The results of evaluating EMT status categorised 13 CRC cell lines as epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. Cytokine/chemokine antibody arrays showed a significant increase in CCL5/RANTES in induced DLD-SIP1 cells. ELISA, multiplex assays and RT-PCR confirmed the increased secretion of CCL5/RANTES in the induced DLDSIP1 cells. The CRC cell line panel showed that the average level of secreted CCL5/RANTES from mesenchymal CRC cells was significantly more than in epithelial cells (639.7 ± 175 vs 107.6 ± 30 pg/ml, respectively; p=0.0075). mRNA expression profiling confirmed this finding from the CRC panel. Promoter studies showed that ZEB1/2 bind to CCL5 promoter, thus activating CCL5 gene expression. No metastatic spread for of DLD-1 cells overexpressing CCL5/RANTES was observed when orthotopically injected into SCID mice. Our data shows that CCL5/RANTES is up-regulated by EMT-inducing transcription factor SIP1, and mesenchymal (metastatic) CRC cells secrete significantly more CCL5/RANTES compared to epithelial (non-metastatic) cells. Furthermore, abundant secretion of CCL5/RANTES might be a crucial regulator of immune infiltration in CRC, but not a direct inducer of metastasis, and that needs to be futher investigated. Inhibiting CCL5 activity in metastatic CRC may have a therapeutic potential.
Supervisor: Bullock, Marc Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available