Context-dependent modulation of aggressiveness of pediatric tumors by individual oncogenic RAS isoforms

Julia Bauer,Katja Simon-Keller,Heidi Hahn,Nicole Cuvelier,Nada Ragab,Dominik P Elmer,Albert Rosenberger,Dominik S Botermann,James A Fagin,Walter Schulz-Schaeffer,Anja Uhmann,Fritz Aberger,Frauke Nitzki,Christian Dullin,Dieter Saur,Stefano Biressi,Natalie Geyer,Hans-Ulrich Schildhaus,Thomas A Rando

doi:10.1038/s41388-021-01904-4

Abstract

A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS, KRAS, or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh-driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.

Highlights

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children with poor prognosis [1]
OncRAS mutations can inhibit GLI1/GLI1 expression via the MEK/ERK axis in human embryonal RMS (ERMS) cell lines In order to investigate the impact of oncogenic RAS (oncRAS) mutations on noncanoncial HH/GLI signaling activity in established human sporadic ERMS, the RAS wildtype ERMS cell lines RUCH-2 and TE617.T were stably transduced with pMSCVpuro-HRASG12V, pMSCVpuro-KRASG12V, pMSCVpuro-NRASG12V, or the pMSCVpuro empty vector (HRAS, KRAS, NRAS, or pMSCV, respectively)
Our results show that oncRAS mutations can negatively regulate GLI1 mRNA expression in RUCH-2 and TE617.T (Fig. 1C, D; results for TE617.T H-/NRAS are not significant)

Summary

Introduction

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children with poor prognosis [1]. ERMS is a prototypic RAS-associated pediatric cancer. Individual oncogenic RAS (oncRAS) mutations affecting all three RAS genes (HRAS, KRAS, and NRAS) occur in up to 42% of ERMS [9,10,11]. Patients with Noonan or Costello syndrome, which are caused by activating K-, N- or HRAS germline mutations, respectively, are predisposed to ERMS [13]. Microarray-based data showing that a RAS signature exists together only with signatures from other activated signaling pathways [5] and the fact that oncRAS mutations are found in ERMS only in combination with other mutations and do not lead to ERMS when occuring alone in the mouse [14,15,16,17] favor the “modifier-hypothesis”. OncRAS mutations seem to play a very important role in ERMS pathogenesis their exact role still remains to be clarified

Methods

Results

Conclusion