Non-Genomic actions of Estradiol and 4OH-Tamoxifen in the Murin breast cancer estrogen dependent cell line LM05-E

RAFFO, D; PONTIGGIA, O; BAL DE KIER JOFFÉ, E; SIMIAN, M

Denver

Congreso; AACR annual meeting 100; 2009

AACR

About 70% of breast tumors are positive for estrogen (ER) and progesterone receptor, being Tamoxifen (T) the main endocrine treatment. However, one third of patients that initially respond to this treatment develop resistance. Recent studies have shown that besides the classical mechanism involving the ER as a transcriptionfactor, this receptor is also responsible for membrane initiated rapid signaling. From a spontaneous estrogen (E) dependent mouse mammary tumor, we generated a cell line, LM05-Mix, composed of an epithelial and a fibroblastic cell population that were cloned to generate the LM05-E and LM05-F cell lines, both ER positive. We previously showed that long term treatment with T (10-6M) induces apoptosis of the LM05-E cells, and that this effect is inhibited when the epithelial cells are co-cultured with the fibroblastic cells. The aim of this work was to study the non-genomic effects of E and T on the LM05-E cells, as reflected on cell growth and protein activation, and to investigate the role of the tumor microenvironment on these responses. Western Bolt analysis was used to determine activation of the MAPK/ERKand PI3K/AKT pathways. To study cell growth, 35.000 LM05-E cells were seeded in 12 well plates and starved for two days. Cells were then treated with short pulses of E (10-8M) or T (10-6M), washed 5 times with PBS, cultured in DMEM/F12 with 1% charcoal stripped serum for additional 48-72 hours and counted. One hour treatment with E induced cell proliferation (P 0.01, n=3), and with T cell death (P 0.001, n=3). Shorter treatments with T of 30 and even 15 minutes also showed a significant reduction in cell growth. A 15 minute treatment with E followed by 1 hour co-culture of E and T showed a partial protection, reducing the negative effect of T (P 0.05, n=3). Preincubation of cells with the MAPK/ERK inhibitor PD98059 (10 uM) made no difference on the proliferation experiments (P 0.01, n=3) even though a reduction in phospho-ERK was observed. Pre-treatment with conditioned media (CM) extracted from tumor fibroblasts induced a reduction of the cytotoxic effect of T (P 0.001, n=3). Pretreatment with GM6001 (10 uM), an MMP inhibitor, abolished cell growth induced by E but didn’t influenced the response to T (P 0.01, n=3). Activation of the MAPK/ERK and PI3K/AKT pathway was observed in response to either E or T treatment between 5 minutes and 1 hour. The ERK activation by T lasted longer than the one induced by E. We conclude that there are ERs associated to membrane able to respond to E and T treatment. On the other hand, MMPs seem to be involved in the E signaling pathway but not T, indicating that despite these two drugs bind ER, they have different signaling pathways downstream this receptor. Finally, the results with tumor fibroblast CM further support a relevant role for the tumor microenvironment on the outcome of the response to T. A Susan G. Komen for the Cure grant to M.S. supports this work.