HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Setareh Safavi,Mikael Eriksson,Christoffer Vannas,Sofia Järnum,Pierre Åman,Eva Wessel Stratford,Ola Myklebost,Regine Schneider Stock,Henrik Fagman,Anders Ståhlberg,Zeynep Kalender,Soheila Dolatabadi,Tajana Tesan Tomic,Pernilla Grundevik,Magnus Hansson,Emma Jonasson,Sameer Udhane,Göran Stenman,Alexandra Jauhiainen

doi:10.18632/oncotarget.6336

Abstract

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.

Highlights

Myxoid liposarcoma (MLS) accounts for more than a third of all liposarcoma cases [1]. This entity is characterized by t(12;16) or more rarely t(12;22) chromosome translocations that result in fusions of the transcription factor gene DDIT3 to FUS or to EWSR1 [2,3,4]
Western blot analysis confirmed that epithelial growth factor receptor (EGFR) (130 kDa) and ERBB3 (190 kDa) proteins were expressed in three MLS cell lines and that at least a fraction of these proteins were phosphorylated at their activation specific positions Y1068 and Y1289, respectively (Figure 2B)
Expression of RET in MLS has independently been reported by others and higher expression level has been associated with poor survival [18, 26, 27]

Summary

INTRODUCTION

Myxoid liposarcoma (MLS) accounts for more than a third of all liposarcoma cases [1]. RET can form active heterodimers with the epithelial growth factor receptor (EGFR) [17] and recently the receptor tyrosine kinase MET was reported to form heterodimers with RET in MLS tissues [18]. Such hetero-complex formation and activation of RET may operate in MLS cells as EGFR is strongly expressed in the tumor cells [19]. We analyzed the expression of RET and its ligands in 8 MLS cases and 4 MLS derived cell lines and investigated its potential interaction with other RTK species. HSP90 inhibitors were tested for effects in MLS cell lines and in an MLS xenograft model

RESULTS

DISCUSSION

MATERIALS AND METHODS