Targeting the CALCB/RAMP1-axis inhibits growth of Ewing sarcoma

Abstract

ABSTRACTEwing sarcoma (EwS) is an aggressive cancer caused by chromosomal translocations generating fusions of theEWSR1gene withETStranscription factors (in 85%FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity increases with the number of consecutive GGAA-repeats.Herein, we investigate the role of the secretory neuropeptide CALCB (calcitonin related polypeptide β) in EwS, which signals via the CGRP-(calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2,678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed thatCALCBis specifically and highly overexpressed in EwS. Time-course knockdown experiments showed thatCALCBexpression is tightly linked to that ofEWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that ofCALCBindicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for EWSR1-FLI1 and histone marks from EwS cells demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close toCALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass-spectrometry analyses of supernatants of EwS cell cultures demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediatedCALCBknockdown had no effect on proliferation and clonogenic growth of EwS cellsin vitro, its long-term knockdown decreased EwS growthin vitroandin vivo. Similarly, knockdown ofRAMP1reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the CGRP-receptor comprising RAMP1 reduced growth of EwS.Collectively, our findings suggest thatCALCBis a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1-axis may offer a new therapeutic strategy for inhibition of EwS growth.