Gliotoxin Targets Nuclear NOTCH2 in Human Solid Tumor Derived Cell Lines In Vitro and Inhibits Melanoma Growth in Xenograft Mouse Model.

Rainer Hubmann,Marlies Reiter,Martin Hilgarth,Ulrich Jäger,Wolfgang Sieghart,Dita Demirtas,Peter Valent,Medhat Shehata,Christoph Zielinski,Susanne Schnabl,Mohammad Araghi

doi:10.3389/fphar.2017.00319

Abstract

Deregulation of NOTCH2 signaling is implicated in a wide variety of human neoplasias. The current concept of targeting NOTCH is based on using gamma secretase inhibitors (GSI) to regulate the release of the active NOTCH intracellular domain. However, the clinical outcome of GSI remains unsatisfactory. Therefore we analyzed human solid tumor derived cell lines for their nuclear NOTCH activity and evaluated the therapeutic potential of the NOTCH2 transactivation inhibitor gliotoxin in comparison to the representative GSI DAPT. Electrophoretic mobility shift assays (EMSA) were used as a surrogate method for the detection of NOTCH/CSL transcription factor complexes. The effect of gliotoxin on cell viability and its clinical relevance was evaluated in vitro and in a melanoma xenograft mouse model. Cell lines derived from melanoma (518A2), hepatocellular carcinoma (SNU398, HCC-3, Hep3B), and pancreas carcinoma (PANC1) express high amounts of nuclear NOTCH2. Gliotoxin efficiently induced apoptosis in these cell lines whereas the GSI DAPT was ineffective. The specificity of gliotoxin was demonstrated in the well differentiated nuclear NOTCH negative cell line Huh7, which was resistant to gliotoxin treatment in vitro. In xenotransplanted 518A2 melanomas, a single day dosing schedule of gliotoxin was well tolerated without any study limiting side effects. Gliotoxin significantly reduced the tumor volume in early (83 mm3 vs. 115 mm3, p = 0.008) as well as in late stage (218 mm3 vs. 576 mm3, p = 0.005) tumor models. In conclusion, NOTCH2 appears to be a key target of gliotoxin in human neoplasias and gliotoxin deserves further evaluation as a potential therapeutic agent in cancer management.

Highlights

The highly conserved NOTCH gene family (NOTCH1-4) encodes trans-membrane receptors that regulate embryonic development and adult tissue homeostasis by modulating binary cell fate decisions in response to external signals (Louvi and Artavanis-Tsakonas, 2012; Ntziachristos et al, 2014)
Deregulation of NOTCH2 signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) (Hubmann et al, 2002; Rosati et al, 2009), marginal zone lymphoma (MZL) (Kiel et al, 2012), pancreas carcinoma (Mazur et al, 2010; Zhou et al, 2013; Liu et al, 2017), hepatocellular carcinoma (HCC) (Dill et al, 2013; Hayashi et al, 2015; Huntzicker et al, 2015; Zhu et al, 2015; Wu et al, 2016), bladder cancer (Hayashi et al, 2016), medulloblastoma (Fan et al, 2004), glioblastoma (Yu et al, 2015), and melanoma (Hoek et al, 2004; Kaushik et al, 2014)
We analyzed nuclear extracts from human cell lines derived from melanoma (518A2), HCC (Hep3B, HCC-3, SNU398, Huh7), and pancreas-CA (PANC-1) for DNA-bound NOTCH/CSL complexes on a CSL consensus site (GTGGGAA) spanning probe by Electrophoretic mobility shift assays (EMSA)

Summary

INTRODUCTION

The highly conserved NOTCH gene family (NOTCH1-4) encodes trans-membrane receptors that regulate embryonic development and adult tissue homeostasis by modulating binary cell fate decisions in response to external signals (Louvi and Artavanis-Tsakonas, 2012; Ntziachristos et al, 2014). Deregulation of NOTCH2 signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) (Hubmann et al, 2002; Rosati et al, 2009), marginal zone lymphoma (MZL) (Kiel et al, 2012), pancreas carcinoma (pancreas-CA) (Mazur et al, 2010; Zhou et al, 2013; Liu et al, 2017), hepatocellular carcinoma (HCC) (Dill et al, 2013; Hayashi et al, 2015; Huntzicker et al, 2015; Zhu et al, 2015; Wu et al, 2016), bladder cancer (Hayashi et al, 2016), medulloblastoma (Fan et al, 2004), glioblastoma (Yu et al, 2015), and melanoma (Hoek et al, 2004; Kaushik et al, 2014) It has been recently shown, for instance, that the nuclear NOTCH2 activity is functionally linked with the self-renewing capacity (stemness) and severity of liver cancer cells making nuclear NOTCH2 an ideal candidate for therapeutic interventions (Zhu et al, 2015). The secondary metabolite gliotoxin was identified as major virulence factor in Aspergillosis patients with immunosuppressive functions and since the discovery of its structure in 1958 (Bell et al, 1958), it became a target for extensive investigations to explore its complex mechanism of action and its multiple downstream effector molecules and for potential drug development (Gardiner et al, 2005; Dolan et al, 2015; Scharf et al, 2016)

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