Three-dimensional (3D) overlay culture models of human breast epithelial malignant progression reveal a critical dependence on MAP kinase activation for Ras-driven proliferation

B106 Although Ras mutations are common in many human cancers, they are rare in breast cancer. Nevertheless, functional Ras activation is prevalent in human breast cancers, presumably due to over-expressed growth factors and their receptors, and there is evidence that therapeutics directed to inhibit the Ras pathway may be useful in breast cancer treatment. We have screened drugs that inhibit several pathways that may be downstream of Ras activation, including MAP kinase, PI 3-Kinase, Rac1 and Rho/myosin light chain kinase (MLCK) for their effects in both traditional, two-dimensional (2D) cell culture and overlay 3D cultures in reconstituted basement membrane (rBM). We tested the agents against: control MCF10A cells, which are a spontaneously immortalized human breast epithelial cell line that retains a normal phenotype by most criteria; a progression series from MCF10A that is termed NeoT, AT1, and DCIS cells (and which model hyperplasia, atypical hyperplasia, and ductal carcinoma in situ in xenograft studies, respectively); and transformed MCF10A that express high levels of activated H-Ras or N-Ras. We used spheroid size to measure cell growth in 3D overlay culture, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to determine proliferation in 2D culture. The MAP kinase kinase inhibitor U0126 potently blocked proliferation of the MCF10.H-Ras and MCF10.N-Ras cells in 3D overlay culture, with EC50 values of 2.1 and 3.7 μM, respectively. A second MAP kinase kinase inhibitor, PD184352, was significantly more potent. Interestingly, U0126 had almost no effect on control MCF10A cells, or the NeoT or AT1 variants, in 3D overlay culture, while having a moderate effect on the MCF10.DCIS cells at the highest concentration tested (10 μM). Western analysis showed that DCIS cells express higher levels of H-Ras protein than the MCF10A, NeoT, and AT1 cells, but less than that in MCF10.H-Ras cells. Remarkably, the selective effect of the MAP kinase kinase inhibitors to inhibit the proliferation of the more transformed cells that was observed in the 3D overlay cultures was not seen in 2D cultures, where 10 μM U0126 produced a similar degree of inhibition of proliferation (30-70%) in all the cell lines tested. In addition to activation of MAP kinase, Ras signaling may lead to other pathways, including activation of PI3K, Rac1 or Rho/MLCK, that could further contribute to malignant progression. In this study, we found no significant growth inhibition in the 3D overlay cultures in response to wortmannin (200 nM, a PI3 kinase inhibitor), NSC23766 (75 μM, a Rac1 inhibitor) or ML-7 (10 μM, a MLCK inhibitor). MTT assays showed only slight effects of these inhibitors in 2D-culture. The results from this study suggest that 3D overlay culture may be a good model for the screening of Ras pathway inhibitors. Using this model, our results indicate that MAP kinase kinase inhibition may be an effective means to selectively suppress Ras-driven proliferation and malignant transformation of breast epithelial cells. Unlike 2D culture, where there was little selectivity for action against the more transformed variants, 3D overlay culture apparently reveals that dependence on MAP kinase activation increases during progression.