Abstract PD3-15: Novel cFlip inhibitor suppresses chemotherapy-induced breast cancer stem cell activity through blocking HIF1-α
Authors: T Robinson, D Turnham, A Gruca, L Piggott, R Clarkson
Journal: Cancer Research (2018)

Abstract Background: The emergence of the cancer stem cell (CSC) hypothesis has helped to explain previously poorly understood clinical concepts such as metastases, late tumour recurrence and resistance to chemotherapy. Triple Negative Breast Cancer (TNBC) has the worst prognosis of all types of breast cancer with a more frequent relapse rate and reduced length of survival in metastatic disease. It has been shown to contain a higher proportion of CSCs than other types of breast cancer. Paclitaxel, a taxane in widespread use in breast cancer, induces apoptosis in a ligand-independent manner through the extrinsic apoptosis pathway. cFLIP is both an antagonist of this apoptosis pathway and has the ability to form aggregates that interfere with the ubiquitylation and subsequent degradation of both HIF1α and β-catenin- two molecules involved in CSC-signalling. Using a novel compound targeted against cFLIP, we wanted to assess whether its combination with paclitaxel effectively targeted CSCs, particularly in TNBC. Methods: In vitro experiments were used to assess the effect on cell viability, mammosphere formation and ALDH+ of both chemotherapy and our novel agent, OH14. We then used siRNA-mediated knockdown of cFLIP to confirm an on-target effect. Annexin V assays were used to assess apoptosis and both Western blotting and qPCR was used to examine the effect of paclitaxel and OH14 on protein levels and gene expression of CSC-signalling pathways. Athymic mice were used to in serial dilution experiments and for in vivo treatmenst with paclitaxel and OH14 on a TNBC cell line. Results: We established an in vitro model demonstrating that a wide range of chemotherapeutic agents (FEC, Paclitaxel and Docetaxel) increased CSC-like behaviour and the ALDH+ population in a broad range of breast cancer cell lines. A mathematical model demonstrated that chemotherapy increased the absolute number of CSCs after treatment suggesting that CSC-like signaling was being induced. OH14, our novel compound, sensitised Triple Negative Breast Cancer (TNBC) cell lines to paclitaxel by increasing its apoptotic effect but had a more profound effect on mammosphere formation and ALDH positivity. This suggests that it preferentially targets CSCs. Serial dilution experiments demonstrated that chemotherapy increased the tumour forming potential of a TNBC cell line in vivo and that this effect was abrogated with OH14. Treatment of the same cell line in vivo demonstrated that paclitaxel +/- OH14 successfully targeted tumours but that the paclitaxel tumours recurred whereas those subject to combined treatment remained undetectable. Further experiments examining CSC signalling showed that HIF1α-mediated signalling was increased by paclitaxel and abrogated by the addition of OH14. Conclusion: cFLIP has a dual effect in both increasing apoptosis and targeting signaling in TNBC CSCs. In a breast cancer subtype in desperate need of novel therapeutic strategies, targeting cFLIP warrants further investigation and progression towards clinical trials. Citation Format: Robinson T, Turnham D, Gruca A, Piggott L, Clarkson R. Novel cFlip inhibitor suppresses chemotherapy-induced breast cancer stem cell activity through blocking HIF1-α [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-15.