Microenvironmental Factors Modulate Tamoxifen Resistance in Breast Cancer.
Authors: O. Pontiggia, D. Raffo, M. Simian
Journal: Cancer Research (2009)

Abstract The tumor microenvironment plays a critical role in tumor growth and progression. However, its impact on response to endocrine therapy has not been fully addressed in the literature. From the estrogen-dependent M05 mouse mammary tumor we established a bicellular cell line, LM05 MIX, which is composed of an epithelial and a fibroblastic cell population which were separated to generate the LM05-E and LM05-F cell lines respectively. Previous work demonstrated that the LM05-E cells, which are positive for estrogen and progesterone receptors, are sensitive to 4-OH-tamoxifen (TAM; 10-6M) being this effect reverted when cells are co-cultured with the fibroblastic LM05-F cell population. Our working hypothesis is that extracellular matrix components and soluble factors produced by the fibroblastic cells may induce tamoxifen resistance on the otherwise sensitive epithelial LM05-E tumor cells. Immunofluorescence staining for the extracellular matrix component laminin (LN) was found to be associated to the fibroblastic cells in mixed cultures. To investigate whether LN conferred TAM resistance, LM05-E cells were cultured on recombinant LN and treated for 48 hours with TAM. We found that LN conferred resistance (P≤ 0.001) to TAM treatment. We used the specific inhibitors PD98059 (ERK ½ inhibitor, 10μM) and LY294002 (PI3K inhibitor, 10μM) to determine which signaling pathways were involved. Only LY294002 reverted TAM resistance, implicating the participation of the PI3K/AKT but not the ERK pathway. Next, to study whether soluble factors secreted by LM05-F cells were mediating TAM resistance we tested the effect of their conditioned media on LM05-E cells in the presence of TAM. We found a decreased number of apoptotic cells in conditioned media-treated cells, as compared to cells treated with non-conditioned media. Treatments with AG1478 (EGFR inhibitor, 6.4 μM), GM6001 (MMP inhibitor, 10 μM), PD98059, LY294002 and AIIB2 (β1-integrin blocking antibody) were used to elucidate which pathways were involved in the protective effect conferred by the conditioned media. The resistance to TAM treatment was reverted with AG1478, GM6001, AIIB2 and LY294002 (P≤ 0.001) but not with PD98059. MMPs or membrane-associated MMP activities have been reported to activate EGFR by shedding EGFR ligands from plasma membrane. Moreover activation of β1-integrin by fibroblast-conditioned media, as suggested by the detection of FAK phosphorylation by western blot, might collaborate with the EGFR signaling pathway. Our results suggest that TAM resistance may be the result of the modulation of epithelial cell responses by tumor fibroblasts via microenvironmental cues such as extracellular matrix components and soluble factors. Targeting the interaction of breast cancer cells with the microenvironment could be a novel and future alternative for patients with breast cancer whose tumors progress to TAM resistance. This work is supported by a grant from the Susan G. Komen for the Cure Foundation to MS (BCTR0600341). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5133.