Fig 4. RKIP inhibits breast cancer metastasis by decreasing MMP13 expression by targeting the Erk2 signaling pathway.

RKIP inhibits breast cancer metastasis by targeting the Erk2 signaling pathway. (a) Heatmap shows changes in expression of MMP13 mRNA in 4T1 cells due to the expression of constitutively active IKK2, Erk2, Akt1 or Stat3 cDNA. Actin mRNA level was used as internal control. The assays were done in triplicate and repeated 3X with similar results. (b) Ectopic expression of RKIP inhibits FBS stimulated activation phosphorylation of Erk1/2.RKIP expressing or control 4T1 cells were serum starved and stimulated with 20% FBS for 5 min. Treated cells were harvested for Western blotting with the indicated Ab. (c) Silencing of RKIP expression enhances FBS stimulated activation phosphorylation of Erk1/2.168FARN RKIP knockdown or control knockdown cells were serum starved and stimulated with 20% FBS for 5 min. Treated cells were harvested for Western blotting with the indicated Ab. (d) The MEK inhibitor U0126 but not the NF-?B inhibitor DHMEQ inhibits 4T1 cell invasion in vitro. Invasion of control and RKIP expressing 4T1 cells through Matrigel in the presence or absence of 50 uM U0126, a MEK inhibitor, or 10 uM DHMEQ, an NF-κB inhibitor, was evaluated. The values represent the means and SEM for the number of cells invading for three wells from three independent experiments. (e-f) Effect of Erk1 or Erk2 expression on RKIP-mediated inhibition of cancer cell invasion. (e) Western blot of Erk1 or Erk2 expression in 4T1 cells infected with the indicated retroviral expression vectors (f) invasion through Matrigel. (g- h)Erk2 expression reverses RKIP-mediated inhibition of lung metastasis. (g) Representative gross lung from mice orthotopically injected with various 4T1 cell lines. (h) Number of metastasis nodules in lungs removed from mice injected orthotopically with 4T1 cells stably infected with the indicated retroviral expression vectors.