Cyclin E-Mediated Human Proopiomelanocortin Regulation as a Therapeutic Target for Cushing Disease

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, 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.

Conclusion: 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.

Figures

Figure 1.. Inhibition of ACTH expression by R-roscovitine in human pituitary corticotroph tumors.

A, Primary cultures of human corticotroph tumor (numbers 1–6) cells treated with vehicle or R-roscovitine for 48 hours. Medium ACTH concentrations in primary cultures were measured by RIA (normalized for viable cell numbers; n = 6 tumors, mean ± SE). *, P < .05. B, POMC mRNA measured by RT-PCR in extracts derived from human corticotroph tumor (numbers 2, 4, 5, and 6) cells treated with vehicle or R-roscovitine. C, Western blot analysis of ACTH expression in primary cultures of human corticotroph tumor (from patient 5 in Table 1) treated with vehicle or R-roscovitine. Tumor numbers correspond to patient numbers in Table 1. RT-PCR was performed in triplicates.

Figure 2.. R-roscovitine inhibition of rPomc promotor activity and corticotroph transcriptional factors.

A, Luciferase assay was performed using rat POMC gene proximal promoter constructs in AtT20 cells treated with or without R-roscovitine under indicated concentrations. B, Luciferase assay of rat Pomc gene promoter construct with Tpit and Pitx1 binding sites mutation in AtT20 cells treated with or without R-roscovitine under the indicated concentrations. Left panel, DNA sequence of the Tpit/Pitx1 binding site is shown. Tpit and Pitx1 sites are indicated in bold. Mutations are indicated by italic capital letters for Tpit and Pitx1 binding sites. C, Western blot of protein extract derived from AtT20 cells treated with vehicle or R-roscovitine. Data shown are representative results from at least two independent experiments.

Figure 3.. R-roscovitine inhibition of cyclin E-mediated regulation of human POMC gene.

A, Western blot of protein extract derived from AtT20 cells transfected with control siRNA (c) or cyclin E siRNA (cyclin E). Representative results from two independent experiments are shown. B, Tpit expression measured by RT-PCR of cDNA derived from human corticotroph tumors numbers 4–6 (Figure 1A) treated with vehicle or R-roscovitine. Each RT-PCR was performed in triplicate and also repeated for tumors 4 and 5. #, Levels are too low to be detected.

Figure 4.. Reciprocal regulation of cyclin E/E2F1 expression mediates Tpit suppression by R-roscovitine.

A, Western blot of protein extracts derived from AtT20 cells treated with vehicle or R-roscovitine. B, POMC and Tpit expression measured by RT-PCR of cDNA derived from AtT20 cells transfected with control siRNA (scrambled) or E2F1 siRNA. C, Western blot of protein extract derived from AtT20 cells transfected with control siRNA (c) or E2F1 siRNA (E2F1). D, Western blot of protein extract derived from AtT20 cells transfected with control siRNA (c), cyclin E (cyclin E), or E2F1 siRNA (E2F1). For illustrative comparison, E2F1 and cyclin E expression from the same Western blot experiments in Figures 3A (cyclin E siRNA) and 4C (E2F1 siRNA) are depicted side by side. CE, cellular protein extract; NE, nuclear protein extract. Data shown are representative results from at least two independent experiments.

Figure 5.. Inhibition of E2F1 interaction with POMC regulatory sequences by R-roscovitine in corticotroph tumors.

ChIP assays were performed in AtT20 cells treated with vehicle (Ros−) or R-roscovitine (Ros+) using PCR primer sets corresponding to the enhancer (ChIP1), the distal (ChIP2), and proximal (ChIP3) promoter region (A) with anti-E2F1 (E2F1) (B), antiacetyl histone H4 (AcH4) (C) antibodies, or negative control (IgG).