Targeting the CALCB/RAMP1 axis inhibits growth of Ewing sarcoma

Abstract

Ewing sarcoma (EwS) is an aggressive cancer characterized by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity correlates 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 2678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated 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 FLI1 and histone marks from EwS cell lines demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass spectrometric analyses of EwS cell culture supernatants demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the RAMP1-comprising CGRP receptor reduced growth of EwS. Collectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1 axis may offer a new therapeutic strategy for inhibition of EwS growth.

CGRP Signaling via CALCRL Increases Chemotherapy Resistance and Stem Cell Properties in Acute Myeloid Leukemia

“…The expression of CGRP, the closely related peptide CGRP2, and CALCRL was increased in some tumor types compared to the corresponding healthy tissues [3,6,7]. CGRP stimulated proliferation and inhibited apoptosis of both normal and malignant cells [3,6,8,9,10,11], and promoted migration and invasiveness of some carcinoma cell lines [3]. Furthermore, CGRP may foster tumor growth through its ability to promote angiogenesis [11].…”

“…Furthermore, CGRP may foster tumor growth through its ability to promote angiogenesis [11]. Accordingly, knockdown of CALCB (which encodes CGRP2 and is activated by the Ewing sarcoma associated fusion protein EWSR1-FLI) or RAMP1 decreased growth of Ewing sarcoma cell lines in vitro and in a mouse xenograft model, and the small molecule CGRP antagonists MK-3207 and olcegepant reduced colony and sphere formation by Ewing sarcoma cells [6].…”

DNA methylation landscape of triple-negative ductal carcinoma in situ (DCIS) progressing to the invasive stage in canine breast cancer

“…Genes involved in transport such as ATOX1, a copper chaperone protein that functions as an antioxidant and is involved in breast cancer cell migration 41,42 and FXYD2, a sodium/potassium-transporting ATPase subunit whose increased expression in tumors may contribute to angiogenesis 43 , were hypomethylated in TN-DCIS and invasive breast cancer. Hypomethylation of signal transduction-related genes BDNF and CALCB have the potential to lead to a myriad of cellular responses associated with cancer such as cell growth and proliferation, angiogenesis, and inflammation 44,45 . Genes involved in pathways related to increased cell motility, PFDN1 and MADCAM1, were hypomethylated while a gene associated with increased cell adhesion, COL7A1, was hypermethylated in TN-DCIS and invasive stages of TNBC.…”

Calcitonin gene‑related peptide: A promising bridge between cancer development and cancer‑associated pain in oral squamous cell carcinoma (Review)

“…A number of studies have revealed that CGRP can directly modulate development of various types of cancer, with a large amount of literature reporting the expression of the CGRP receptor complex in cancer cells (55)(56)(57). CGRP was demonstrated to be able to modify the chemokinetic abilities of a metastatic breast cancer cell line and increase the expression of its receptors (58).…”

“…In addition, it has been demonstrated that CGRP increased the invasive ability of prostate cancer PC-3 cells and an osteosarcoma cell line (13,50). Recently, Dallmayer et al (56) revealed that targeting the CALCB/RAMP1 (the receptor complex of β-CGRP) axis inhibited the growth of Ewing sarcoma cells. Despite the lack of studies on the role of CGRP in OSCC, several studies have revealed a wide distribution of CLR/RAMP1 in oral soft tissues, bones and dental tissues (59).…”