Abstract
IntroductionIt is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs, however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal.MethodsSuppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation.ResultsGenetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment.ConclusionsWe describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.
Highlights
It is postulated that breast cancer stem cells mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients
While 10% to 30% of the cancer cell populations remained viable after Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)/FLIP inhibition (FLIPi) treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional breast cancer stem cells (bCSCs) pool, eliminating stem cell renewal
To determine if the targeted inhibition of cellular FLICE-Like Inhibitory Protein (c-FLIP) exhibited similar specificity for tumour cells, mammary epithelial cell viability was assessed in non-tumourgenic c-FLIP-deficient mouse mammary glands, transformed murine cell lines and in the human breast cell line MCF-10A. c-FLIP was conditionally deleted from mammary epithelial cells of juvenile mice by crossing the blg-Cre transgene [43] into the cFLIPfl/fl line [42], and the mammary epithelial compartment subsequently assessed in adult virgin and pregnant animals
Summary
It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Recognition that breast cancer is a heterogeneous disease has helped shape advances in therapy, leading to more targeted therapeutic strategies and improved survival rates in discrete disease subgroups [1] This is exemplified by the advent of therapeutic agents targeting estrogen-receptor positive (ER+) and HER2-positive (HER2+) breast cancers, which make up approximately 70% of all breast tumours [2,3]. At the centre of this problem lies additional tumour heterogeneity whereby a small population of cells within, or possibly outside, the tumour are both resistant to drugs and provide the source of new tumour growth [4,5] These cells contribute directly to the seeding of secondary tumours at distal sites, the primary cause of mortality in breast cancer patients [6]. There is, considerable interest in targeting CSCs within tumours with cytotoxic agents as a cure for breast and other cancers and where possible to broaden the specificity of therapeutic agents to treat as wide a patient group as possible
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