Breast tumours have intrinsic heterogeneity. The cancer stem cell hypothesis is currently challenging the notion that all cancer cells are equally malignant, suggesting that it is important to evaluate the efficacy of potential anti-cancer drugs by their ability to target the stem-like population. TRAIL is a cytotoxic agent the efficacy of which has been limited by a lack of patient stratification in clinical trials (Lemke 2014). In pre-clinical studies TRAIL has shown specificity towards mesenchymal-like breast cancer cell lines (Rahman et al. 2009) We show here that TRAIL is able to target the tumoursphere-forming population of four out of six breast cancer cell lines, including two epithelial-like lines, the bulk population of which is TRAIL-resistant. Furthermore, TRAIL also reduced the tumour-initiating capacity of the MCF-7 line. In addition, we have also investigated a paracrine mechanism of sensitising breast cancer cell lines to TRAIL. We have shown that a soluble factor produced by MDA-MB-231 cells, fibroblasts, and cancer-associated fibroblasts (CAFs) can sensitise both MCF-7 cells and SKBR3 tumoursphere-forming cells to TRAIL. Our data shows that cytoplasmic levels of Cellular FLICE-Like Inhibitory Protein (cFLIP) – a naturally occurring inhibitor of TRAIL’s cell toxicity effects- are lower in TRAIL-sensitive cells and suggest that tumoursphere populations are TRAIL-sensitive due to the re-localisation of cFLIP to the nucleus. We believe cFLIP is nuclear in stem-like cells due to a role as a promoter of the Wnt pathway. We have shown that inhibition of cFLIP by siRNA resulted in a reduction in both beta-catenin protein levels and Wnt-target gene transcription in both the MCF-7 and MDA-MB-231 breast cancer cell lines. We have also demonstrated a novel role for cFLIP as a promoter of bCSC maintenance. We have found that inhibition of cFLIP by shRNA decreased the self-renewal of tumoursphere-forming cells and also reduced colony formation. As TRAIL alone does not completely eradicate tumoursphere-forming or tumour-initiating cells in any breast cancer cell line, we believe our data are evidence of bCSC heterogeneity existing in terms of susceptibility to TRAIL. We propose a model of phenotypic heterogeneity within breast cancer cell lines and bCSCs whereby there exist two populations of cells which can be distinguished based on TRAIL susceptibility correlating with the known distinction of epithelial-like or mesenchymal-like status and our novel observation of cFLIP localisation. While these findings are currently restricted to cell lines, if confirmed in primary breast cancer cells, the clinical implication of our model is that although TRAIL alone is a potential therapy, a much more effective therapeutic strategy would be to also inhibit cFLIP, the consequences of which would not just be a sensitisation to TRAIL but also a reduction in Wnt signalling, and potentially a reduction in bCSC self-renewal and proliferation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:633536 |
| Date | January 2014 |
| Creators | French, Rhiannon |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/68201/ |
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