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 5

Nuclear EGFR localization in corticotroph adenomas.

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Nuclear EGFR localization in corticotroph adenomas. (A) Confocal immunoc...
(A) Confocal immunocytochemistry for EGFR was performed in human and canine corticotroph tumors. Human breast cancer tissue was used as a positive control, and human testis as negative control, for EGFR expression. Original magnification, ×100. (B–E) AtT20 cells were transiently transfected with EGFR (pCMV/EGFR/myc/nuc), ICD (pCMV/EGFR-ICD/myc/nuc), or EV (pCMV/myc/nuc) for 24 hours. (B) Immunofluorescent chemistry was performed with anti-EGFR. Original magnification, ×100. (C) Media were changed to serum-depleted media and collected 24 hours later. ACTH levels in culture media were measured using RIA (normalized for cell numbers). (D) RNA was extracted, and mouse Pomc expression levels were measured by real-time PCR. A representative experiment is shown (normalized by Gapdh). (E) After gefitinib treatment for 24 hours, RNA was extracted, and mouse Pomc expression levels were measured by real-time PCR. A representative experiment is shown (normalized by Gapdh). Values are mean ± SEM. *P < 0.05, **P < 0.01, vs. EV. Representative results are from triplicate samples in at least 2 independent experiments.