Abstract P5-03-15: Epithelial-to-mesenchymal transition; what is the impact on breast cancer stem cells and drug resistance
Authors: Mallini P , Lennard T , Kirby J , Meeson A 
Published: August 2014

Introduction: SP cells are a subpopulation of Cancer Stem Cells (CSCs) which have been isolated from many types of tissue, including the breast. SP cells can be sorted due to their ability to expel Hoechst 33342 dye through members of ATP Binding Cassette (ABC) transporters superfamily of proteins. This property could explain their ability to also export cytotoxic drugs leading to drug resistant tumors. Epithelial-to-Mesenchymal Transition (EMT) is a process known to contribute to metastasis in cancer via the TGF-β signalling pathway and it mainly results in the downregulation of E-cadherin. Conversely, BMP-7 is a known inducer of the reverse process (MET), leading to inhibition of invasiveness and metastasis. TGF-β1 treatment in the breast cancer cell line MCF-7 eliminated the SP population, decreased the levels of ABCG2 expression and reduced cell viability in the presence of mitoxantrone. The role of BMP-7 on SP cells has not been explored. In this study, we investigated the effects of TGF-β1 treatment on SP cells of the MDA-MB-231 cell line. Autocrine production of TGF-β in the MDA-MB-231 cells is thought to be essential for their growth and maintenance. Therefore, we examined the effects of exogenous TGF-β1 and BMP-7 treatment on the SP cells.

Methods: We have utilised FACS combined with Hoechst 33342 dye efflux to identify cells with an SP phenotype in the MDA-MB-231 cell line. Moreover, using standard techniques, including Real-Time PCR and ICC we determined the expression of TGF-β receptor genes (TGFB-RI and TGFB-RII) and the activation of the TGF-β signalling pathway (Smad 2/3 phosphorylation) when TGF-β1(5 ng/ml) is added to MDA-MB-231 cell cultures. Cultures were also treated with BMP-7 (100 ng/ml) and the impact on p-smad 1/5/8 was examined by ICC. Finally, we treated the total cell population with TGF-β1 and BMP-7 for 3 days and performed SP analysis.

Results: The MDA-MB-231 cell line contains an SP population and it expresses receptors for TGF-β1. Successful activation of the corresponding signaling pathways is achieved as confirmed by the detection of p-smad 2/3 and p-smad 1/5/8 after TGF-β1 and BMP-7 treatment, respectively. TGF-β1 led to the depletion of the SP cells, whereas these effects were reversed upon the addition of the TGFB-RI antagonist, SB-505124. Interestingly, treatment with SB-505124 in the absence of TGF-β1 significantly increased the percentage of SP cells (p = 0.0285), but it also inhibited the cell growth of the total population. BMP-7 enhanced the SP numbers and this was confirmed by the use of the BMP-RI inhibitor, LDN193189.

Conclusions: The presence of TGF-β1 leads to loss of the SP cell phenotype in the MDA-MB-231 cells, while BMP-7 has the opposite role. Definition of the exact EMT and MET mechanisms that regulate this subpopulation could enable the identification of new potential therapeutic targets and the development of more efficient strategies for the management of metastasis and drug resistance in breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-15.