EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas
Hidenori Fukuoka, … , Dave Bruyette, Shlomo Melmed
Hidenori Fukuoka, … , Dave Bruyette, Shlomo Melmed
Published November 21, 2011
Citation Information: J Clin Invest. 2011;121(12):4712-4721. https://doi.org/10.1172/JCI60417.
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Research Article

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas

Abstract

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease.

Authors

Hidenori Fukuoka, Odelia Cooper, Anat Ben-Shlomo, Adam Mamelak, Song-Guang Ren, Dave Bruyette, Shlomo Melmed

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Figure 4

Effects of gefitinib on corticotroph adenoma cell proliferation.

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Effects of gefitinib on corticotroph adenoma cell proliferation. (A) EGF...
(A) EGFRWT, L858R, or EV cells were treated with gefitinib (0.1–10 μM) for 24 hours, and cells were counted. (B) EGFRWT cells were seeded (4,000 cells/well) for colony-forming assay, and gefitinib (0.1–10 μM) was added with serum-depleted media every third day. Colonies were counted from 5 randomly selected fields. (C) EGFRWT cells were treated with gefitinib (0.1–10 μM) for 48 hours, and TUNEL staining was performed. Original magnification, ×20. Apoptotic cells were counted from 5 randomly selected fields (normalized by DAPI-stained cell number). (D) EGFRWT cells were treated with gefitinib (0.1–10 μM) for 48 hours, and Western blotting was performed. (E) EGFRWT, L858R, or EV cells were treated with gefitinib (0.1–10 μM) for 24 hours, BrdU staining was performed, and incorporation was quantified using flow cytometry. (F) EGFRWT cells were treated with gefitinib (0.1–10 μM) for 24 hours with or without EGF (5 nM), and Western blotting was performed. Values are mean ± SEM. #P < 0.01, ##P < 0.01 vs. untreated control; §P < 0.001 vs. vehicle control; **P < 0.01 vs. EV control; †P < 0.05, ††P < 0.01 vs. EGFRWT control; ‡P < 0.05, ‡‡P < 0.01 vs. L858R control. Representative results are from triplicate samples in at least 2 independent experiments.