Abstract P4-02-11: Treatment with Fibronectin or Carcinoma Associated Fibroblast Conditioned Media Generate Sustained Tamoxifen Resistance in Breast Cancer Cells

Abstract

Abstract Background: breast cancer is the most common neoplasm in woman and the second cancer-related cause of death. About 70% of breast cancers are positive for estrogen receptor (ER) and progesterone receptors. Tamoxifen is the main hormonal therapy for ER+ patients. However, one third of the patients that are initially responsive eventually develop resistance to this treatment. We previously showed that treatment with fibronectin or conditioned media from carcinoma associated fibroblasts conferred tamoxifen resistance to otherwise sensitive ER+ breast cancer cells (in particular MCF-7 and LM05-E cells). On the other hand we also found that in the case of conditioned media, the PI3K/AKT pathway was involved downstream of EGFR and ≥1 integrin, whereas for fibronectin both the MAPK/ERK1/2 and the PI3K/AKT pathways were involved downstream of ≥1. The aim of this study was to investigate whether the protective effect conferred by the stromal elements was reversible, or, if on the contrary, it was sustained over time even after they were removed from the culture. In order to do so we pre-treated LM05-E cells with DMEM/F12 containing 1% charcoal stripped serum (CSS) as a control or with the addition of fibronectin (10μg/ml) or conditioned media from the LM05-F carcinoma associated fibroblast cell line. Subsequently cells were trypsinized, washed and allowed to attach ON in 8 well chamber slides in the absence of the stromal factors. They were then treated for 48 hours with estradiol 10-8 M, or estradiol plus 4-OH-tamoxifen 10-6 M in the presence of 1% CSS. Cell death was measured by propidium iodide incorporation. The 48 hour pre-treatment with either conditioned media or fibronectin conferred resistance to tamoxifen induced cell death. This resistance was sustained for at least a week in both cases, as determined by culturing the cells for this period of time in the absence of the stromal factors before testing the effect of 4-OH-tamoxifen. Shorter pretreatments of 6 or 12 hours weren't capable of conferring endocrine resistance. Inhibitors of the MAPK/ERK, PI3K/AKT or EGFR were added to the pre-treatments in order to study involvement of these pathways. Neither addition of the PI3K inhibitor LY294002 (10 μM), the MAPK inhibitor PD98059 (10 μM) or the EGFR inhibitor AG1478 (6,4 μM) were able to reverse the sustained protection induced by either fibronectin or conditioned media. Conclusion: our results suggest that both of these stromal elements are able to induce sustained changes in the breast cancer cells that lead to tamoxifen resistance even after they have been removed from the culture. At least a 48 hour period is required to accomplish this protection and this effect is sustained for at least a week. These results have important clinical implications on the lasting effects that the tumor microenvironment may have on the long term response to endocrine treatment in breast cancer. This work is supported by a grant from the Susan G. Komen for the Cure Foundation to MS (BCTR0600341). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-11.