Hedgehog Pathway Inhibition Hampers Sphere and Holoclone Formation in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal (eRMS) and alveolar (aRMS). Among the cellular heterogeneity of tumors, the existence of a small fraction of cells called cancer stem cells (CSC), thought to be responsible for the onset and propagation of cancer, has been demonstrated in some neoplasia. Although the existence of CSC has been reported for eRMS, their existence in aRMS, the most malignant subtype, has not been demonstrated to date. Given the lack of suitable markers to identify this subpopulation in aRMS, we used cancer stem cell-enriched supracellular structures (spheres and holoclones) to study this subpopulation. This strategy allowed us to demonstrate the capacity of both aRMS and eRMS cells to form these structures and retain self-renewal capacity. Furthermore, cells contained in spheres and holoclones showed significant Hedgehog pathway induction, the inhibition of which (pharmacologic or genetic) impairs the formation of both holoclones and spheres. Our findings point to a crucial role of this pathway in the maintenance of these structures and suggest that Hedgehog pathway targeting in CSC may have great potential in preventing local relapses and metastases.

Sarcoma treatment in the era of molecular medicine

“…The expression of these factors in sarcomas is oncogene‐driven and triggered by a combination of mutational and epigenetic events or by developmental programs (Rodriguez et al , 2012; Xiao et al , 2013). These events ultimately result in the deregulation of pathways that control stemness and differentiation, such as Hedgehog, Notch, Wnt/β‐Catenin, Hippo, or ALK (Graf Finckenstein et al , 2008; Naka et al , 2010; Riggi et al , 2010; Rodriguez et al , 2013; Basu‐Roy et al , 2015, 2016; Eid & Garcia, 2015; Tamaki et al , 2015; Abarrategi et al , 2016; Almazán‐Moga et al , 2017; Slemmons et al , 2017; Deel et al , 2018; Genadry et al , 2018; Hatina et al , 2019; Rodríguez‐Núñez et al , 2019; Schiavone et al , 2019; Trautmann et al , 2019). Alternatively, both stemness and aggressiveness can be regulated by the interaction with cells in the TME (Alfranca et al , 2015; Schiavone et al , 2019), or physical and chemical properties of the TME (i.e., hypoxia and extracellular acidosis) (Zeng et al , 2011; Alfranca et al , 2015; Avnet & Cortini, 2016; Avnet et al , 2017).…”

Soft Tissue Sarcoma Cancer Stem Cells: An Overview

“…On the other hand, the genomic heterogeneity of ERMS highlights the challenge of finding a single target for therapeutic purposes. Using a variety of approaches, cell populations with CSC features have been reported for ERMS ( 49 – 52 ); the identification of ARMS CSCs has been more elusive and while a recent study showed that ARMS cells could form holoclones and spheres ( 53 ), no studies have reported in vivo functional assays for ARMS CSCs. Similar to what is observed in SS [below ( 54 )], there is some thought that almost all PAX3-FOXO1 + ARMS tumor cells have stem cell characteristics–suggesting that ARMS is a stemness-disease, but this has yet to be demonstrated.…”

“…Each sphere consists of a small percentage of self-renewing cells and a large percentage of progenitor cells at various stages of differentiation ( 133 ). In the last few years, sphere culture techniques have been employed to allow CSC enrichment in STSs, including both ERMS and ARMS ( 50 , 53 ), FS ( 77 ), SS ( 54 , 77 ), MPNSTs ( 94 ), LMS ( 105 ), and LPS ( 118 , 119 ).…”

“…The HH pathway has been proven to be hyperactivated also in ARMS, although it appears mainly activated in translocation-negative ARMS compared to translocation-positive ones ( 202 ), and its upregulation has been associated with a poor prognosis ( 204 ). A recent study showed HH pathway activation in cells contained in CSC-enriched structures including holoclones and spheres ( 53 ). In UPS, the HH pathway has been linked to the maintenance of self-renewal and proliferation of a subpopulation of putative CSCs characterized by a SP phenotype ( 126 ).…”

Genetics, epigenetics and redox homeostasis in rhabdomyosarcoma: Emerging targets and therapeutics

“…Thus ERMS containing GLI1 and NANOG seem to have clinically worse outcome [92]. HH pathway was also shown to be induced in cancer stem cell enriched spheres and holoclones of both ERMS and ARMS cell types [93]. IHH, DHH and SHH ligand expression in RMS activates HH signalling in an autocrine manner.…”