Abstract
Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3beta (GSK-3beta). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3beta activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-beta (PDGFRbeta) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3beta in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3beta and of nuclear cyclin D1.
Highlights
Transitional cell carcinoma (TCC) of the bladder is the fourth most common solid tumor malignancy in the United States
Through studies done on a panel of 10 bladder cancer cells, we found that epidermal growth factor receptor (EGFR) blockade suppresses cell proliferation in a subgroup of cell lines in a manner that was not tightly linked to the receptor’s expression levels
We characterized a panel of 10 human urothelial carcinoma cell lines for EGFR, HER2, and HER4 expression, as well as pEGFR, and found that all the cell lines tested expressed various levels of HER2 and EGFR protein and that some cell lines displayed higher levels of activated EGFR (Fig. 1A)
Summary
Transitional cell carcinoma (TCC) of the bladder is the fourth most common solid tumor malignancy in the United States. The uncoupling between Ras/MAPKs and EGFR activation in the resistant UM-UC13 cell line suggests that the ability of EGFR inhibitors to exert their antiproliferative effect was dependent upon the balance between the activation status of MAPK and Akt, which could be reflected by the activation www.aacrjournals.org of a common downstream kinase, such as GSK-3h.
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