Sarcomas induced in discrete subsets of prospectively isolated skeletal muscle cells

Abstract

Soft-tissue sarcomas are heterogeneous cancers that can present with tissue-specific differentiation markers. To examine the cellular basis for this histopathological variation and to identify sarcoma-relevant molecular pathways, we generated a chimeric mouse model in which sarcoma-associated genetic lesions can be introduced into discrete, muscle-resident myogenic and mesenchymal cell lineages. Expression of Kirsten rat sarcoma viral oncogene [Kras(G12V)] and disruption of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16p19) in prospectively isolated satellite cells gave rise to pleomorphic rhabdomyosarcomas (MyoD-, Myogenin- and Desmin-positive), whereas introduction of the same oncogenetic hits in nonmyogenic progenitors induced pleomorphic sarcomas lacking myogenic features. Transcriptional profiling demonstrated that myogenic and nonmyogenic Kras; p16p19(null) sarcomas recapitulate gene-expression signatures of human rhabdomyosarcomas and identified a cluster of genes that is concordantly up-regulated in both mouse and human sarcomas. This cluster includes genes associated with Ras and mechanistic target of rapamycin (mTOR) signaling, a finding consistent with activation of the Ras and mTOR pathways both in Kras; p16p19(null) sarcomas and in 26-50% of human rhabdomyosarcomas surveyed. Moreover, chemical inhibition of Ras or mTOR signaling arrested the growth of mouse Kras; p16p19(null) sarcomas and of human rhabdomyosarcoma cells in vitro and in vivo. Taken together, these data demonstrate the critical importance of lineage commitment within the tumor cell-of-origin in determining sarcoma histotype and introduce an experimental platform for rapid dissection of sarcoma-relevant cellular and molecular events.

Fig. 1.

Tumor initiation by Kras(G12V)-expressing p16p19^null MFA cells. (A) Mouse MFA cell subsets are discriminated by combinatorial staining for CD45, MAC1, Sca1, β1-integrin and CXCR4. (B) Differentiation of satellite cells (blue gate in A) and a subset of CXCR4⁻ cells (purple gate in A) into myosin heavy chain-positive (MYHC⁺) myoblasts and myofibers (B, Upper). Differentiation of Sca1⁺ cells (red gate in A) and a subset of CXCR4⁻ cells (purple gate in A) into Oil-Red-O⁺ adipocytes (B, Lower). (C and D) p16p19^null MFA cells were infected with Kras(G12V) lentivirus and injected into the cardiotoxin-preinjured gastrocnemius muscles of NOD/SCID mice 36–48 h after cell isolation. Kaplan-Meier analysis (C) showed no differences in the percent of mice developing tumors (D) induced by individual cell types (P = 0.8). (E) Limiting dilution analysis showed equivalently high frequencies of tumor-initiating cells within each of the individual subsets of Kras(G12V)-transduced p16p19^null MFA cells (P = 0.4). FSC, Forward Scatter; SSC, Side Scatter; Pi, Propidium Iodide; MAC1, Macrophage-1 antigen; TER119, antigen recognized by anti-Ly76 antibody; Sca1, stem cell antigen-1; CXCR4, C-X-C chemokine receptor type 4; Beta1, β1 integrin.

Fig. 2.

Morphological and IHC evaluation of tumors arising from Kras(G12V)-expressing p16p19^null cells. H&E (Top row) and IHC staining of Kras(G12V);p16p19^null tumors for MyoD (Second row), myogenin (MyoG) (Third row), desmin (Fourth row), GFP (Fifth row), and Ki67 (Bottom row). Representative images are shown for a MyoD-, myogenin-, and desmin-positive sarcoma arising from satellite cells (A), a MyoD- and myogenin-negative sarcoma arising from CXCR4⁻ cells (B), and a MyoD- and myogenin-negative sarcoma arising from Sca1⁺ cells (C). Images were obtained at 20× magnification. (Scale bars, 100 μm.) Boxed areas are shown at threefold greater magnification at right.

Fig. 3.

Transcriptional profiling of Kras; p16p19^null mouse sarcomas. (A–C) GSEA analysis (details are given in Table S2) to test for enrichment of the set of Kras; p16p19^null sarcoma-associated genes by comparing (A) embryonal RMS (ERMS), (B) Paired box 3-Forkhead Transcription Factor–positive (P3+) alveolar RMS (ARMS), and (C) non-RMS STS with normal muscle. The upper quartile of genes (A–C, marked in gray) identified by GSEA analyses shows 144 overlapping genes (194 overlapping probes), which are coordinately regulated in human RMS and non-RMS STS. (Fig. S3B and Table S3). (D–F) GSEA analysis demonstrates enrichment of this cluster of overlapping genes in (D) MCF10a cells transduced with Ras, (E) HEK cells transduced with Ras, and (F) HMECs transduced with Ras compared with nontransduced control cells but not in HMECs transduced with other oncogenes (Table S4).

Fig. 4.

Ras/Raf/MEK/ERK and mTOR signaling regulate the growth of Kras; p16p19^null sarcomas and the human RMS cell line RD. (A–C) Proliferation of two stable cell lines established from mouse Kras; p16p19^null RMS tumors of satellite-cell origin (A), of two stable cell lines established from mouse Kras; p16p19^null nonmyogenic sarcomas of Sca1⁺ origin (B), and of the human RMS cell line RD (C) was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in control cultures (dark blue in A and red in B) and after inhibiting mTOR by exposure to 50–100 nMol rapamycin (abbreviated R, light blue in A and orange in B) or to 50–100 nMol torin (abbreviated T) or by inhibiting Ras/Raf/MEK/ERK by exposure to 1–10 μMol U0216 (abbreviated U). (D–G) Mice with Kras; p16p19^null RMS tumors of satellite-cell origin (D and F) or Kras; p16p19^null nonmyogenic sarcomas of Sca1⁺ origin (E and G) were treated daily with rapamycin; treatment prolonged time until killing (P < 0.0001) and impeded growth of both myogenic and nonmyogenic sarcomas.