BRD4 inhibitor IBET upregulates p27kip/cip protein stability in neuroendocrine tumor cells

Abstract

The prevalence of neuroendocrine tumors (NETs) has recently been increasing. Although various drugs such as Octreotide and its analogs show certain efficacy, NETs in many patients progress and metastasize. It is desirable to develop new interventions to improve the therapy. Here we show that human neuroendocrine tumor BON cells are resistant to several drugs commonly used for NET therapy, including Octreotide that activates somatostatin receptor-induced anti-proliferation, and Capecitabine and Temozolimide that damage DNA. In contrast, an inhibitor (IBET) to an epigenetic regulator, Brd4 that binds acetylated histones and upregulates transcription of multiple genes including protooncogene c-Myc, potently inhibited the NET cells. We found that IBET increased the protein levels of cyclin-dependent kinase (CDK) inhibitor p27^kip/cip (or p27), but not its mRNA levels. Moreover, the p27 induction at protein level by IBET was at least partly through increasing the protein stability of p27. The increased protein stability of p27 likely resulted from IBET-mediated suppression of Skp2, an E3 ligase that can mediate p27 degradation by increasing its ubiquitinylation. These findings unravel a new mechanism whereby the IBET-induced repression of proliferation of neuroendocrine cells.

Keywords: Brd4; IBET; Skp2; neuroendocrine tumor; p27.

Figure 1.

The effect of various drugs on BON cell growth. (A-D) BON cells were treated with various concentrations of everolimus (A), octreotide (B), capecitabine (C) and temozolimide (D). MTS assay was performed to assess cell proliferation after 3 d. (E) BON cells were treated with varying concentrations of IBET and MTS assay was performed to assess cell proliferation after 5 d of treatment. (F) BON cells were treated with various concentrations of Foxoi and cell counting was performed to assess cell proliferation after 5 d of the treatment.

Figure 2.

The time course of treatment of BON cells with various drugs. (A) Cell counting following treatment of the BON cells with IBET (500nM) for the indicated time. (B) Cell counting following treatment of the BON cells with everolimus (100nM) for the indicated time. (C) Cell counting following treatment of the BON cells with Foxoi(100nM) for the indicated time. (D) Cell counting following treatment of the BON cells with capecitabine (5μM) for the indicated time.

Figure 3.

The effect of IBET treatment on the protein levels of p27, FOXO1 and c-Myc. The BON cells were treated with control DMSO or IBET (500 nM), for 5 days, followed by Western blotting analysis using the indicated antibodies.

Figure 4.

The effect of IBET treatment on the mRNA level of p27cip/kip. BON cells were treated with either control DMSO or IBET (500 nM) for 5 days, followed by isolation of RNA from the cells, and qRT-PCR analysis for the FOXO1 and p27 (B). The means of the different groups in the mRNA level were compared using one-way ANOVA test. Differences were considered statistically significant when P < 0.05(*P < 0.05).

Figure 5.

IBET treatment increases p27 protein stability. (A) BON cells were treated with DMSO or IBET for 3 days, followed by addition of 20 μg/ml cycloheximide in the respective medium, and the cells were collected at 0, 2, 4 and 6 hr after the cycloheximide treatment of Western blotting with the indicated antibodies. (B) p27 protein band in (A) was scanned, and was quantitated using Image J and plotted as % protein relative to the signal value at 0 min.

Figure 6.

The effect of IBET and FOXOi treatment on the protein level of Skp2. (A) BON cells were treated with either DMSO or IBET (0.5uM) for 3 days, followed by Western blotting analysis to detect the protein levels with the indicated antibodies. (B) BON cells were treated with DMSO or FOXOi (100nM) for 3 days, followed by Western blotting with the indicated antibodies. (C) A working model to explain IBET-induced upregulation of p27 protein level via suppressing expression of Skp2.