Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population

Abstract

BackgroundThe identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD).MethodsRhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium.ResultsMEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells.ConclusionsOur results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance.Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0501-y) contains supplementary material, which is available to authorized users.

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

“…Furthermore, GLPG1790 was able to radiosensitize ERMS cells by affecting the radiation-induced activation of the DNA-repair mechanisms. At molecular levels, GLPG1790 inhibited MAPK and AKT signal transduction pathways, two well-established major drivers of ERMS oncogenicity [22][23][24][25]. Similar effects were obtained by silencing EPH-A2 and/or EPH-B2, the principal EPH receptors targeted by GPLG1790 [21], this suggesting the specificity of GLPG1790 activity.…”

“…Our data also suggest that the GLPG1790-mediated acquisition of the myogenic-like phenotype and the enhanced expression of specific myogenic genes, such as MYOD1, myogenin and MyHC, seems to pass through the reduction of DNMT3B protein levels, whose knockdown has recently been shown to be a key event in reactivating the ERMS terminal differentiation program [28]. According to our recent report that shows how the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of ERMS cells [22], we found that GLPG1790 dramatically prevented rhabdosphere formation and downregulated the expression of the stem cell markers CD133, CXCR4 and Nanog. The finding that GLPG1790 treatment induces cell cycle alteration and commits ERMS cells to myogenesis suggests that this compound has a therapeutic potential as a differentiating agent in ERMS tumours.…”

“…In particular, in both ERMS cell lines, GLPG1790 concomitantly reduced activation status of AKT, mTOR, ERK, JNK and Src proteins, whose related signalling are known to promote ERMS development and to block terminal muscle differentiation [22,23,[41][42][43][44]. Notably, p38 phosphorylation levels were dramatically increased by GLPG1790 treatment, confirming the key role of this mitogen-activated protein kinase in the reactivation of the terminal differentiation program in both RD and TE671 cells [22,28,[41][42][43][44]. Our data also suggest that the GLPG1790-mediated acquisition of the myogenic-like phenotype and the enhanced expression of specific myogenic genes, such as MYOD1, myogenin and MyHC, seems to pass through the reduction of DNMT3B protein levels, whose knockdown has recently been shown to be a key event in reactivating the ERMS terminal differentiation program [28].…”

“…The biological mechanisms altered by GLPG1790 include the modulation of several signal transduction pathways, which are involved in promoting oncogenic transformation and progression in many types of tumours, and which have been previously linked to EPH RTKs [6][7][8]. In particular, in both ERMS cell lines, GLPG1790 concomitantly reduced activation status of AKT, mTOR, ERK, JNK and Src proteins, whose related signalling are known to promote ERMS development and to block terminal muscle differentiation [22,23,[41][42][43][44]. Notably, p38 phosphorylation levels were dramatically increased by GLPG1790 treatment, confirming the key role of this mitogen-activated protein kinase in the reactivation of the terminal differentiation program in both RD and TE671 cells [22,28,[41][42][43][44].…”

“…DNA profiling using the GenePrint 10 System (Promega Corporation, Madison, WI) was carried out to authenticate cell cultures, comparing the DNA profile of our cell cultures with those found in GenBank. Sphere-forming cells were obtained and radiation treatment performed as already described [22]. EPH receptor inhibitor GLPG1790 was provided by Galapagos NV [21], dissolved in DMSO and stored at − 20°C until use.…”