Effects of epigenetic pathway inhibitors on corticotroph tumour AtT20 cells

Abstract

Medical treatments for corticotrophinomas are limited, and we therefore investigated the effects of epigenetic modulators, a new class of anti-tumour drugs, on the murine adrenocorticotropic hormone (ACTH)-secreting corticotrophinoma cell line AtT20. We found that AtT20 cells express members of the bromo and extra-terminal (BET) protein family, which bind acetylated histones, and therefore, studied the anti-proliferative and pro-apoptotic effects of two BET inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, using CellTiter Blue and Caspase Glo assays, respectively. JQ1 and PFI-1 significantly decreased proliferation by 95% (P < 0.0005) and 43% (P < 0.0005), respectively, but only JQ1 significantly increased apoptosis by >50-fold (P < 0.0005), when compared to untreated control cells. The anti-proliferative effects of JQ1 and PFI-1 remained for 96 h after removal of the respective compound. JQ1, but not PFI-1, affected the cell cycle, as assessed by propidium iodide staining and flow cytometry, and resulted in a higher number of AtT20 cells in the sub G1 phase. RNA-sequence analysis, which was confirmed by qRT-PCR and Western blot analyses, revealed that JQ1 treatment significantly altered expression of genes involved in apoptosis, such as NFκB, and the somatostatin receptor 2 (SSTR2) anti-proliferative signalling pathway, including SSTR2. JQ1 treatment also significantly reduced transcription and protein expression of the ACTH precursor pro-opiomelanocortin (POMC) and ACTH secretion by AtT20 cells. Thus, JQ1 treatment has anti-proliferative and pro-apoptotic effects on AtT20 cells and reduces ACTH secretion, thereby indicating that BET inhibition may provide a novel approach for treatment of corticotrophinomas.

Preclinical drug studies in MEN1-related neuroendocrine neoplasms (MEN1-NENs)

“…Endocrine-Related Cancer pancreatic, pituitary and bronchial NEN cell lines, as well as reducing ACTH secretion from the ACTH-secreting pituitary cell lines, AtT20 (Lines et al 2017(Lines et al , 2020. Furthermore, in vivo, assessment using a pancreatic β-cellspecific conditional Men1-knockout mouse model that develops pNENs, revealed that JQ1 decreased proliferation and increased apoptosis of pNENs (Lines et al 2017).…”

The bromodomain inhibitor JQ1+ reduces calcium-sensing receptor activity in pituitary cell lines

“…Increasing evidence indicates that aberrant epigenetic modifications play an important role in pituitary tumourigenesis ( Shariq & Lines 2019 ), and previously we have demonstrated that the bromo and extra terminal domain (BET) protein inhibitor, JQ1+, reduces proliferation and increases apoptosis of the corticotroph murine cell line, AtT20 ( Lines et al 2020 ), indicating it may also represent an effective novel therapy for corticotrophinomas. The mechanism by which JQ1+ alters proliferation and apoptosis in these cells is, however, incompletely understood.…”

“…The mechanism by which JQ1+ alters proliferation and apoptosis in these cells is, however, incompletely understood. To elucidate the target pathways of JQ1+ in pituitary cells, we previously undertook RNA sequence analysis ( Lines et al 2020 ). This revealed that one of the most highly significantly downregulated genes was the calcium-sensing receptor (CaSR) (Supplementary Table 1, see section on supplementary materials given at the end of this article).…”

“…Reduction of CaSR expression by siRNA has been shown to reduce CaSR signalling in AtT20 cells, although the effect on proliferation was not assessed ( Mamillapalli & Wysolmerski 2010 ). Recently, JQ1+ treatment of AtT20 cells was shown to reduce proliferation and increase apoptosis, indicating that this may be an efficacious treatment for corticotrophinomas ( Lines et al 2020 ). RNA-Seq analysis in these JQ1+-treated cells revealed that the Casr gene was significantly downregulated ( Lines et al 2020 ) (Supplementary Table 1).…”

“…Recently, JQ1+ treatment of AtT20 cells was shown to reduce proliferation and increase apoptosis, indicating that this may be an efficacious treatment for corticotrophinomas ( Lines et al 2020 ). RNA-Seq analysis in these JQ1+-treated cells revealed that the Casr gene was significantly downregulated ( Lines et al 2020 ) (Supplementary Table 1). We, therefore, hypothesised that reducing CaSR expression or signalling by the receptor may be a novel treatment for pituitary tumours.…”