Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Austin Proudfit,Neetu Gupta,Debasis Pore,Yvonne Parker,Nabanita Bhunia,Daniel Lindner

doi:10.1155/2020/9010496

Abstract

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

Highlights

Rhabdomyosarcoma (RMS) is regarded as the most common pediatric soft tissue sarcoma with an incidence of 4.4 cases per million annually [1]. ese tumors are of mesenchymal origin and occur in diverse anatomical locations, including the head, neck, trunk region, within the genitourinary tract, and abdominal area [2,3,4,5].e two major histological subtypes of RMS are embryonal (ERMS) and alveolar (ARMS), with Embryonal rhabdomyosarcoma ARMS (ERMS) diagnosed more commonly (∼60% cases) compared to ARMS (∼20% cases) [6]
Given that phosphorylated-ezrin ( r567)-radixin ( r564)-moesin ( r558) levels determine the degree of activity in cells, the phosphorylation status of these proteins was examined in two ERMS subtype (RD and Rh18) and two ARMS subtype (Rh30 and Rh41) cell lines derived from different subtypes
Western blotting revealed that ERM proteins were constitutively phosphorylated at regulatory threonine residue in all RMS cell lines, regardless of subtype or fusion status (Figure 1(a))

Summary

Introduction

E two major histological subtypes of RMS are embryonal (ERMS) and alveolar (ARMS), with ERMS diagnosed more commonly (∼60% cases) compared to ARMS (∼20% cases) [6]. ERMS typically presents in younger children (1–14 yrs.) relative to ARMS; both major subtypes are most often diagnosed as pediatric sarcomas [7, 8]. Frequent genetic alterations in ERMS include loss of heterozygosity (LOH) and imprinting at the 11p15.5 locus, Sarcoma resulting in the overexpression of IGF2 [11,12,13]. Risk classification of RMS is based on multiple criteria including histological subtype, tumor location, and metastatic potential [6]. Standard treatment regimens for RMS include combination chemotherapy along with surgery and/or radiation, resulting in a five-year event free survival rate of ∼90% for localized, low-risk patients. The estimated survival rates drop to ∼70% in patients with intermediate and ∼25% high-risk RMS, underscoring a need for novel therapeutic options [15,16,17]

Objectives

Methods

Results

Conclusion