HuR-targeted small molecule inhibitor exhibits cytotoxicity towards human lung cancer cells

Ranganayaki Muralidharan,Rajagopal Ramesh,Terence Herman,Jeffrey Aubé,Rebaz Ahmed,Meghna Mehta,Sudeshna Roy,Anupama Munshi,Yan Daniel Zhao,Liang Xu,Allshine Chen

doi:10.1038/s41598-017-07787-4

Abstract

Human antigen (Hu) R is an RNA-binding protein whose overexpression in human cancer correlates with aggressive disease, drug resistance, and poor prognosis. HuR inhibition has profound anticancer activity. Pharmacologic inhibitors can overcome the limitations of genetic inhibition. In this study, we examined the antitumor activity of CMLD-2, a small-molecule inhibitor directed against HuR, using non-small cell lung cancer (NSCLC) as a model. CMLD-2 efficacy was tested in vitro using H1299, A549, HCC827, and H1975 NSCLC cells and MRC-9 and CCD-16 normal human fibroblasts. Treatment of NSCLC cells with CMLD-2 produced dose-dependent cytotoxicity, caused a G1 phase cell-cycle arrest and induced apoptosis. CMLD-2 decreased HuR mRNA and the mRNAs of HuR-regulated proteins (Bcl2 and p27) in tumor cells. Additionally, reduction in the expression of HuR, Bcl2, cyclin E, and Bcl-XL with increased expression of Bax and p27 in CMLD-2-treated NSCLC cells were observed. CMLD-2-treated normal cells, HuR-regulated mRNAs and proteins albeit showed some reduction were less compared to tumor cells. Finally, CMLD-2 treatment resulted in greater mitochondrial perturbation, activation of caspase-9 and -3 and cleavage of PARP in tumor cells compared to normal cells. Our proof-of concept study results demonstrate CMLD-2 represents a promising HuR-targeted therapeutic class that with further development could lead to advanced preclinical studied and ultimately for lung cancer treatment.

Highlights

HuR is an RNA-binding protein that regulates the stability and transcription of numerous mRNAs whose protein products function as oncoproteins and are frequently overexpressed in several human cancers, including lung cancer[1,2,3]
CMLD-2 is a coumarin-derived molecule that was identified through fluorescence polarization (FP) assay-based high throughput screening (HTS) of a library of 6,000 compounds developed by the Kansas University Chemical Methodologies and Library Development (CMLD) center[31]
Initial studies were focused on testing the cytotoxic concentration of CMLD-2 that was effective in suppressing tumor cell growth

Summary

Introduction

HuR is an RNA-binding protein that regulates the stability and transcription of numerous mRNAs whose protein products function as oncoproteins and are frequently overexpressed in several human cancers, including lung cancer[1,2,3]. These studies utilized anti-sense oligonucleotide or small interfering (si) RNA to inhibit HuR While these results established proof-of-concept, there are several barriers, such as poor cell uptake and low serum stability, to siRNA-based therapy. Consistent with our results, other laboratories have reported similar treatment outcomes with HuR siRNA11–14, 28 While all of these studies are promising, advancing siRNA-based therapy to the clinic remains a challenge. Concurring with these findings, Romeo et al.[30], showed MS-444 treatment reverted TRAIL resistance in pancreatic cancer Both studies provide evidence that small molecule inhibitors can successfully disrupt HuR and produce anticancer activity. While HuR-targeted small molecule inhibitors such as CMLD-2 have been developed and tested in pancreatic and colon cancers, their efficacy against human lung cancer cells is not known

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Results

Conclusion