Abstract and Introduction
Abstract
Introduction: 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. 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.
Methods: Suppression 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.
Results: Genetic 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.
Conclusions: We 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.
Introduction
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. 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. Despite these improvements, however, tumours often relapse due to innate or acquired resistance to the therapeutic insult. 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. These cells also contribute directly to the seeding of secondary tumours at distal sites, the primary cause of mortality in breast cancer patients. These drug resistant cancer initiating cells, often referred to as breast Cancer Stem Cells (bCSCs), have been demonstrated functionally for both human and mouse mammary tumours and tumour cell lines. Experiments on human breast tumours in mouse models, for example, indicate that when these cells were deleted, the remaining cells were unable to sustain new tumour growth. There is, therefore, 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.
Tumour Necrosis Factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) is a promising anticancer agent that exhibits tumour specificity with only mild side effects observed in clinical trials for the treatment of colorectal cancer, non-small cell lung carcinoma and non-Hodgkins lymphoma. In breast cancer, however, its therapeutic potential is limited by the fact that the majority of breast cancer cell types are resistant to TRAIL. This has prompted much interest in identifying agents that might increase TRAIL sensitivity in a larger cohort of breast cancer patients. Moreover, stem cells, including cancer stem cells, are documented to be resistant to TRAIL, suggesting that without further sensitization of the tumour-initiating cell sub-population, patients are likely to relapse following TRAIL therapy.
TRAIL targets tumour cells for instructive cell death via the cell-surface receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which initiate the formation of death inducing signalling complexes (DISCs) ultimately leading to the activation of the caspase cascade. A number of studies have described agents that sensitize one or more breast cancer subtypes to TRAIL, the majority of which implicate components of the apoptosis regulatory machinery as the underlying causes of sensitization. Common to a number of these studies is the observation that the endogenous inhibitor of death receptor killing, cellular FLICE-Like Inhibitory Protein (c-FLIP), is down-regulated during the sensitization process. c-FLIP is a non-redundant antagonist of caspases -8 and -10, preventing these caspases from binding to the DISC and thus inhibiting autolytic cleavage and subsequent activation of downstream executioner caspases following stimulation by TRAIL. The suppression of c-FLIP has been shown to sensitize some breast cancer cell lines to TRAIL mediated killing, raising the possibility that such a mechanism could be targeted in breast cancer patients. However, several questions concerning the specificity of c-FLIP in breast cancer remain that would significantly impact on its prospects as a therapy for breast cancer. These include: whether suppressing c-FLIP in non-tumour cells compromises their viability; whether a broad range of breast cancer subtypes are affected by c-FLIP sensitization; and of particular clinical significance, whether the normally chemo-resistant CSC sub-populations within each of these heterogeneous subtypes are sensitive to de-repression of this apoptotic pathway.
Here we addressed each of these clinically relevant questions by selectively targeting c-FLIP in pre-clinical models of breast cancer. We looked at the effects of suppressing c-FLIP in non-tumourgenic cells, and showed that c-FLIP exhibited tumour cell specificity, similar to that previously ascribed to TRAIL in other tumour types. Moreover, we demonstrated that the de-repression of TRAIL by c-FLIP inhibition selectively eliminated breast cancer stem cells (bCSCs) from tumour cell populations, irrespective of their HER2/ER receptor status and despite CSC plasticity within the surviving tumour cell population. These observations were then confirmed in in vivo models of breast cancer whereby primary tumourgenesis was reduced by 80% and the seeding of new tumour growth at distal sites, leading to metastatic disease, was almost completely inhibited. These findings demonstrate potent cellular responses to TRAIL sensitization that have important clinical implications for the advent of new therapeutic strategies for breast cancer patients.