Cyclin E-Mediated Human Proopiomelanocortin Regulation as a Therapeutic Target for Cushing Disease.
BACKGROUND
Cushing disease, due to pituitary corticotroph tumor ACTH hypersecretion, drives excess adrenal cortisol production with adverse morbidity and mortality. Loss of glucocorticoid negative feedback on the hypothalamic-pituitary-adrenal axis leads to autonomous transcription of the corticotroph precursor hormone proopiomelanocortin (POMC), consequent ACTH overproduction, and adrenal hypercortisolism. We previously reported that R-roscovitine (CYC202, seliciclib), a 2,6,9-trisubstituted purine analog, suppresses cyclin-dependent-kinase 2/cyclin E and inhibits ACTH in mice and zebrafish. We hypothesized that intrapituitary cyclin E signaling regulates corticotroph tumor POMC transcription independently of cell cycle progression. The aim was to investigate whether R-roscovitine inhibits human ACTH in corticotroph tumors by targeting the cyclin-dependent kinase 2/cyclin E signaling pathway.


METHODS
Primary cell cultures of surgically resected human corticotroph tumors were treated with or without R-roscovitine, ACTH measured by RIA and quantitative PCR, and/or Western blot analysis performed to investigate ACTH and lineage-specific transcription factors. Cyclin E and E2F transcription factor 1 (E2F1) small interfering RNA (siRNA) transfection was performed in murine corticotroph tumor AtT20 cells to elucidate mechanisms for drug action. POMC gene promoter activity in response to R-roscovitine treatment was analyzed using luciferase reporter and chromatin immunoprecipitation assays.


RESULTS
R-roscovitine inhibits human corticotroph tumor POMC and Tpit/Tbx19 transcription with decreased ACTH expression. Cyclin E and E2F1 exhibit reciprocal positive regulation in corticotroph tumors. R-roscovitine disrupts E2F1 binding to the POMC gene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways.


CONCLUSIONS
R-roscovitine inhibits human pituitary corticotroph tumor ACTH by targeting the cyclin E/E2F1 pathway. Pituitary cyclin E/E2F1 signaling is a previously unappreciated molecular mechanism underlying neuroendocrine regulation of the hypothalamic-pituitary-adrenal axis, providing a subcellular therapeutic target for small molecule cyclin-dependent kinase 2 inhibitors of pituitary ACTH-dependent hypercortisolism, ie, Cushing disease.
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Ning-Ai
NA
Liu
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Takako
T
Araki
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Daniel
D
Cuevas-Ramos
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Jiang
J
Hong
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Anat
A
Ben-Shlomo
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Yukiko
Y
Tone
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Masahide
M
Tone
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048.
Shlomo
S
Melmed
D049913:ACTH-Secreting Pituitary Adenoma / Q000150:complications / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D000236:Adenoma / Q000150:complications / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D000293:Adolescent; D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D019927:Cyclin E / Q000037:antagonists & inhibitors* / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D058990:Molecular Targeted Therapy / Q000379:methods*; D047748:Pituitary ACTH Hypersecretion / Q000188:drug therapy* / Q000473:pathology; D061251:Primary Cell Culture; D011333:Pro-Opiomelanocortin / Q000235:genetics* / Q000378:metabolism; D011687:Purines / Q000627:therapeutic use*; D000077546:Roscovitine; D014407:Tumor Cells, Cultured; D055815:Young Adult
D000970:Antineoplastic Agents; D019927:Cyclin E; D011687:Purines; D000077546:Roscovitine; D011333:Pro-Opiomelanocortin