Cyclin e-mediated human proopiomelanocortin regulation as a therapeutic target for cushing disease

Ning Ai Liu, Takako Araki, Daniel Cuevas-Ramos, Jiang Hong, Anat Ben-Shlomo, Yukiko Tone, Masahide Tone, Shlomo Melmed

Research output: Contribution to journalArticle

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Abstract

Context: 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,ACTHmeasuredby RIAandquantitative 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 thePOMCgene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibitshumanpituitary corticotroph tumorACTHby 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.

Original languageEnglish (US)
Pages (from-to)2557-2564
Number of pages8
JournalJournal of Clinical Endocrinology and Metabolism
Volume100
Issue number7
DOIs
StatePublished - Jul 1 2015

Fingerprint

Corticotrophs
Pituitary ACTH Hypersecretion
Pro-Opiomelanocortin
Cyclins
E2F1 Transcription Factor
Cyclin E
Adrenocorticotropic Hormone
Tumors
Cyclin-Dependent Kinase 2
Transcription
Neoplasms
Therapeutics
Cushing Syndrome
roscovitine
Primary Cell Culture
Chromatin Immunoprecipitation
Pituitary Neoplasms
Zebrafish
Luciferases
Cell culture

Cite this

Cyclin e-mediated human proopiomelanocortin regulation as a therapeutic target for cushing disease. / Liu, Ning Ai; Araki, Takako; Cuevas-Ramos, Daniel; Hong, Jiang; Ben-Shlomo, Anat; Tone, Yukiko; Tone, Masahide; Melmed, Shlomo.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 100, No. 7, 01.07.2015, p. 2557-2564.

Research output: Contribution to journalArticle

Liu, Ning Ai ; Araki, Takako ; Cuevas-Ramos, Daniel ; Hong, Jiang ; Ben-Shlomo, Anat ; Tone, Yukiko ; Tone, Masahide ; Melmed, Shlomo. / Cyclin e-mediated human proopiomelanocortin regulation as a therapeutic target for cushing disease. In: Journal of Clinical Endocrinology and Metabolism. 2015 ; Vol. 100, No. 7. pp. 2557-2564.
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abstract = "Context: 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,ACTHmeasuredby RIAandquantitative 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 thePOMCgene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibitshumanpituitary corticotroph tumorACTHby 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.",
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T1 - Cyclin e-mediated human proopiomelanocortin regulation as a therapeutic target for cushing disease

AU - Liu, Ning Ai

AU - Araki, Takako

AU - Cuevas-Ramos, Daniel

AU - Hong, Jiang

AU - Ben-Shlomo, Anat

AU - Tone, Yukiko

AU - Tone, Masahide

AU - Melmed, Shlomo

PY - 2015/7/1

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N2 - Context: 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,ACTHmeasuredby RIAandquantitative 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 thePOMCgene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibitshumanpituitary corticotroph tumorACTHby 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.

AB - Context: 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,ACTHmeasuredby RIAandquantitative 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 thePOMCgene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibitshumanpituitary corticotroph tumorACTHby 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.

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