Figure 3.

MAPK-induced MMP9 expression prevents phenotypic reversion of T4-2 cells induced by multiple inhibitors. (A) Schematic of Raf–MEK–ERK pathway. Ligand-induced activation of RafER bypasses upstream regulators, allowing temporal control of MEK activation. (B) Dose response of T4-2^vector and T4-2^RafER cells. Treatment with increasing concentrations of β-estradiol increases p-Mek-1 in the T4-2^RafER in relation to control cells. Ponceau-stained bands indicate equal loading. (C) Zymography of T4-2^RafER CM. Increasing β-estradiol concentrations leads to increased levels of MMP9. (D) T4-2^vector and T4-2^RafER cells were treated with DMSO, AG1478, LY294002, or GM6001 for 7 d in 3D lrECM cultures and were stained for α6-integrin and DNA. T4-2^vector cells revert similar to wild-type T4-2 cells (top panel), whereas T4-2^RafER cells are resistant to reverting agents (bottom panel). (E) Histogram representing the relative proliferation of T4-2^vector and T4-2^RafER cells determined by WST-1 reagent after 7 d in 3D lrECM. Proliferation of T4-2^vector is significantly reduced by reverting agents, whereas T4-2^RafER remains highly proliferative. (F) Phase-contrast images of T4-2^vector and T4-2^RafER cells treated with DMSO, AG1478, or a higher concentration of GM6001 (80 μM compared with 40 μM used in D for 7 d in 3D lrECM. Cells remain resistant to reversion by AG1478, indicating that RafER is active. Increasing the concentration of GM6001 counteracts the action of activated RafER observed in D and leads to reversion of T4-2^RafER. (G) Immunoblot analysis of total cell lysates from 3D lrECM cultures of T4-2^RafER cells treated with indicated inhibitors. EGFR and p-Mek-1 levels remain elevated even in the presence of the reverting agents. Total Mek levels remain unchanged. Immunoblot of total cell lysates from 3D lrECM cultures of control T4-2^vector is presented in Figure 2A and indicates that phenotypic reversion is accompanied by a decrease in signaling through Mek. Ponceau-stained protein bands indicate equivalent sample loading. (H) Zymography of CM collected from T4-2^RafER cells treated with DMSO, AG1478, Ly294002, or GM6001 in 3D lrECM shows that MMP9 production remains elevated in the presence of the reverting agents due to continued signaling through the MEK pathway. Zymography of CM collected from control T4-2^vector cells is presented in Figure 1D. Loading was normalized to cell number, as determined by WST-1 reagent.