Effects of RAS on the genesis of embryonal rhabdomyosarcoma

Abstract

Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.

Figure 1.

rag2-kRASG12D-injected animals develop RMS. (A) Bright-field image of a 30-d-old zebrafish with RMS. (B) Tumor onset in AB strain fish injected with rag2-kRASG12D (49 of 105 animals developed RMS by 80 dpf). (C–F) RMS cells invade into adjacent muscle (C), intestine (D), liver (E), and kidney (F). Dotted line in D outlines the outer edge of effaced intestine. Arrowheads denote striated muscle tumors. The boxed region in E is a magnified view of a striated cell in the liver. Bars: C–F, 50 μm. (I–T) kRASG12D-induced tumors express clinical diagnostic markers of RMS as determined by RNA in situ hybridization. (I,K,M,O,Q,S) Antisense probes. (J,L,N,P,R,T) Sense controls. Arrowheads in S denote multinucleated, myogenin-positive cells within RMS. Bars: I–T, 20 μm. (U) Semiquantitative RT–PCR comparing normal muscle and RMS from 30-d-old fish. Embryo cDNA served as a positive control in most samples (24 hpf). (mylz2) myosin light chain 2; (ckm) creatine kinase.

Figure 2.

GSEA identifies a conserved gene signature found in both zebrafish and human ERMS. (A) Heat map showing genes up-regulated in zERMS when compared with normal muscle at 2.25-fold change (left) and juxtaposed to the corresponding human orthologs in ERMS, ARMS, and normal juvenile muscle (right). (B–E) Graphical representation of the rank-ordered gene lists found when comparing human RMS to normal muscle. The up-regulated gene set identified in zebrafish RMS is significantly enriched in human ERMS (B; ES = 0.414, NES = 1.518, FDR q-val = 0.023, p = 0.013) but not the alveolar subtype (C; ES = 0.384, NES = 1.251, FDR q-val = 0.223, p = 0.155). The down-regulated gene set identified in zebrafish RMS is not significantly enriched in either ERMS (D) or ARMS (E). The yellow box in B defines the genes that contribute maximally to the GSEA score in human ERMS. (ES) Enrichment score.

Figure 3.

GSEA identifies a novel evolutionarily conserved RAS signature and a tumor-specific signature associated with ERMS. (A) The up-regulated gene set identified in zERMS is significantly enriched in the human ERMS, pancreatic adenocarcinoma, and RAS-infected mammary epithelial cell (HMEC) data sets (denoted by bold lettering), while the down-regulated gene set is not significantly enriched in any data set. (ES) Enrichment score; (NES) Normalized Enrichment Score; (FDR) False discovery rate; (FWER p-val) FWER p-value. The asterisks denote samples that have discordant gene set enrichment, exhibiting up-regulation of a down-regulated gene set. (B) The genes from the up-regulated zERMS gene set that contribute maximally to the GSEA score in the pancreatic adenocarcinoma, human ERMS, and RAS-infected HMEC data sets differ. The 24 genes comprising the TSTR are marked. (C) Previously identified RAS signatures share few genes in common with the up-regulated gene set identified in the zebrafish transgenic model of RAS-induced RMS (2.25-fold change gene list). Genes contained within each overlapping group are noted in B and C.

Figure 4.

p53 pathway deregulation alters tumor onset in zebrafish RAS-induced ERMS. p53 LOF collaborates with kRASG12D to increase penetrance of RMS in animals injected with the rag2-kRASG12D transgene at the one-cell stage of life (wild-type vs. heterozygotes, p = 0.0039; wild-type vs. homozygotes, p < 0.00001; heterozygotes vs. homozygotes, p = 0.00001; n = 98 of 137 homozygous p53 LOF fish developed tumors, n = 103 of 217 in heterozygous fish, and n = 5 of 28 in wild-type fish).

Figure 5.

Coinjection strategies can be used to label distinct cell populations within zebrafish RMS. (A) GFP fluorescent image of RMS developing in a rag2-dsRED2⁺/α-actin-GFP⁺-injected animal. (B) GFP fluorescence in cryostat section. (C) dsRED2 fluorescence image of injected fish shown in A. (D) dsRED2 fluorescence in cryostat section. (E,F) Merged images. Arrowheads in B, D, and F denote cells that expresses GFP (G⁺), dsRED2⁺ (R⁺) or both (R⁺G⁺) within zebrafish RMS. Bars: B,D,F, 20 μm. (G) FACS profile of α-actin-GFP transgenic animal injected at the one-cell stage with rag2-dsRED2 and rag2-kRASG12D. (H–K) The four cell populations can be isolated to relative purity by FACS. (L) Semiquantitative RT–PCR analysis confirms that expression of dsRED2⁺ and GFP⁺ can be used to identify discrete populations of tumor cells based on their stage of muscle differentiation. Total refers to total cells isolated from RMS by FACS based on cell viability. (M) Microarray analysis of sorted cell populations from three tumors (numbered 1–3 at top of heat map). Gene symbols are at right, with blue denoting genes expressed in normal differentiating muscle cells.

Figure 6.

The dsRED2⁺ cell population from double-transgenic rag2-dsRED2/α-actin-GFP animals contains the serially transplantable cancer stem cell in zERMS. (A–D) Primary transplanted tumors from α-actin-GFP⁺/rag2-dsRED2⁺ fish (1° Recipient). (A) Merged image of GFP fluorescent, dsRED2 fluorescent, and brightfield images. (B) FACS analysis of primary recipient engrafted with ERMS. (C,D) Histological analysis reveals heterogeneity in transplant animals, with some fish having masses of spindled cells (C) or round cell aggregates (D), or both. Bars: C,D, 100 μm. (E–G) Cells isolated from serially transplanted animals, in this case a quaternary recipient animal (4° Recipient). (E) FACS plot of tumor cells isolated from a 4° recipient. (F) Wright-Giemsa-stained cytospins of FACS-sorted R⁺ cells from quaternary tumor. (G) Semiquantitative RT–PCR analysis of FACS-sorted cell populations. Total refers to total cells isolated from quaternary transplanted RMS isolated by FACS based on cell viability and that serve as an input control. (H–M) Fish transplanted with 50 R⁺ cells defined in E–G (5° Recipient). (H) Bright-field image of transplant recipient animal. (I) Merged image of a dsRED2⁺/GFP⁺ tumor in same animal. Hematoxylin and eosin-stained (J) and anti-GFP-immunostained (K) section of transplanted fish showing that RMS cells infiltrate the liver (L), head kidney (HK), and skeletal muscle. (L,M) High-power magnification of boxed region in J. Bars: J,K, 1 mm; L,M, 100 μm.