Investigating the therapeutic potential of cellular FLICE-like inhibitory protein and TRAIL in preclinical models of breast cancer

Doctoral thesis English OPEN
Piggott, Luke
  • Subject: Q1 | RC0254 | RM

Apoptosis is an important process in normal mammary gland physiology and evasion of apoptosis has also been identified as a hallmark of cancer. In breast cancer cells apoptotic resistance is an acquired feature that can promote tumour growth and progression. Induction of apoptosis by the extrinsic death ligand TRAIL has been shown to be a promising clinical therapy targeting a number of different cancer cells whilst sparing normal cells. Unfortunately most breast cancers are inherently resistant to TRAIL treatment. Herein it is shown that by reducing the expression of the downstream TRAIL inhibitor c-FLIP, a range of different breast cancer subtypes can be sensitised to TRAIL treatment resulting in significant cancer cell death. Significantly, suppression of c-FLIP in combination with TRAIL (FLIPi/TRAIL) ablated the tumour-initiating breast cancer stem cell (bCSC) subset, as defined by mammosphere formation assay, within cell lines. This selective killing of bCSCs translated to reduced tumour initiation and metastasis in animal transplant models. However, continued culture of FLIPi/TRAIL treated cell lines in adherent conditions resulted in bCSC re-acquisition suggesting a phenotypic plasticity of non-bCSC cells. Re-acquired bCSCs also demonstrated sensitivity to repeated FLIPi/TRAIL treatment and maintaining reduced c-FLIP expression prevented bCSC re-acquisition. These results substantiate the importance of resistance to apoptosis in tumour initiation and metastasis and identify the targeting of c-FLIP proteins as a promising anti-cancer therapeutic approach. \ud Acquired resistance to existing mainstay therapies such as antiestrogens (AEs) (tamoxifen and Faslodex) and aromatase inhibitors (AIs) is an ongoing obstacle in treatment of a large number of breast cancer patients. AE-resistant models of breast cancer and a multiple endocrine-resistant patient sample demonstrated hypersensitivity to TRAIL. This sensitivity was observed in both in vitro and in vivo models of AE resistance and cell death was prevalent in both bulk tumour cells and bCSCs. Sensitisation was not attributed to combination AE/TRAIL treatment suggesting cellular changes during the acquisition of AE resistance are responsible for TRAIL sensitivity in these models. Further investigation suggested that the mechanism of AE-resistant cell sensitivity to TRAIL was not dependant on functional estrogen receptor signalling and is most likely dependant on the AE agent that the cancer cells have acquired resistance to. Interestingly tamoxifen-resistant MCF-7 cells were shown to have reduced c-FLIP protein expression compared to parental cells, further supporting c-FLIP’s potential in cancer therapy. \ud Recent success in the use non-MHC-restricted γδ T cells as a targeted immunotherapy in clinical trials has identified this therapeutic methodology as desirable. Here it is shown that TRAIL is readily expressed by this subset of T cells that also demonstrate cytotoxicity to breast cancer cell lines. Neither the secretion of TRAIL or surface expression of TRAIL appeared to contribute significantly towards γδ T cell cytotoxicity and the majority of breast cancer cell death induced by γδ T cells would seem to be perforin-mediated. The suppression of c-FLIP in target cells increased γδ T cell cytotoxicity but again not via TRAIL. Preliminary results also indicated that the bCSCs of some cell lines were exquisitely sensitive to γδ T cell treatment. In summary these results indicate that targeting c-FLIP and TRAIL can be therapeutically beneficial in a range of different breast cancer subtypes by certain therapeutic strategies.
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