Pharmacological Inhibition of Class IIA HDACs by LMK-235 in Pancreatic Neuroendocrine Tumor Cells.

Julia Wanek,Marlena Beyreis,Tobias Kiesslich,Andrej Wagner,Daniel Neureiter,Florian Primavesi,Martin Gaisberger,Pietro Di Fazio,Tarkan Jäger,Christian Mayr,Martin Jakab,Katharina Helm

doi:10.3390/ijms19103128

Abstract

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

Highlights

Pancreatic neuroendocrine tumors are a heterogeneous group of malignancies originating from cells of the diffuse endocrine system of the pancreas [1]
HDAC5 expression was investigated in several tumor entities and its oncogenic de-regulation has been found to be dependent on the cancer type [13,14]
HDAC5 over-expression has been demonstrated in human pancreatic neuroendocrine tumors (pNETs) tissue samples by Klieser et al [21] and correlated with poor clinical outcome, especially in terms of low overall and disease-free survival

Summary

Introduction

Pancreatic neuroendocrine tumors (pNETs) are a heterogeneous group of malignancies originating from cells of the diffuse endocrine system of the pancreas [1]. Dysfunction and or expression changes of HDACs have been observed in a wide range of tumors, including breast cancer, melanoma, and prostate cancer [10,12,13,14,15,16] This led to the development of the first pharmaceutical HDAC inhibitor (HDACi), which received FDA approval in 2006, and several more being recently approved or being investigated in trials [17,18,19]. In the current study, we analyzed the in vitro effects of pharmacological HDAC inhibition (LMK-235, a selective class IIA HDAC inhibitor [31]) in two established pNET cell lines, BON-1 and QGP-1, to determine whether LMK-235 represents an effective modifier of this epigenetic mechanism and a possible therapeutic approach in pNET

Objectives

Methods

Results

Conclusion