Distinct Malignant Behaviors of Mouse Myogenic Tumors Induced by Different Oncogenetic Lesions

Rhabdomyosarcomas (RMS) are heterogeneous cancers with myogenic differentiation features. The cytogenetic and mutational aberrations in RMS are diverse. This study examined differences in the malignant behavior of two genetically distinct and disease-relevant mouse myogenic tumor models. Kras; p1619null myogenic tumors, initiated by expression of oncogenic Kras in p16p19null mouse satellite cells, were metastatic to the lungs of the majority of tumor-bearing animals and repopulated tumors in seven of nine secondary recipients. In contrast, SmoM2 tumors, initiated by ubiquitous expression of a mutant Smoothened allele, did not metastasize and repopulated tumors in 2 of 18 recipients only. In summary, genetically distinct myogenic tumors in mice exhibit marked differences in malignant behavior.


INTRODUCTION
Rhabdomyosarcomas (RMS) are heterogeneous cancers with myogenic differentiation (1). Fusion-positive RMS tumors carry exclusive chromosomal translocations at t(2;13)(q35;q14) or t(1;13)(p36;q14) and exhibit aggressive clinical behavior (2,3). The remaining, fusion-negative spectrum of human RMS comprises a diverse group of tumors with frequent RAS pathway activation (4,5) and variable mutations, including loss of heterozygosity at the PTCH1 locus (6,7) in a subset of fusion-negative RMS. PTCH1 serves as a Hedgehog (Hh) receptor, and loss of PTCH1 function results in de-repression of downstream Hh pathway signaling. The contributions of RMS-relevant oncogenic pathways, including RAS and Hh signaling, to myogenic tumor formation were previously tested in mice (8,9). This report highlights the distinct phenotypes of two mouse myogenic tumor modelsthose initiated by combined Cdkn2a (p16p19) disruption and Kras expression in transplanted mouse muscle satellite cells (10) and those arising in the skeletal muscle of mice with activated Hh signaling due to expression of a mutant, constitutively active smoothened (SmoM2) allele (11,12). We demonstrate significant differences in tumor-repopulating activity and prevalence of lung metastases between Kras-driven and Hh-driven myogenic tumors in mice. These observations reveal marked differences in malignant behavior between genetically distinct mouse myogenic tumors, suggesting that an understanding of the distinct oncogenetic underpinnings of tumors on the fusion-negative RMS spectrum may be informative for clinical prognosis and treatment.

R26-SmoM2
(mixed genetic background including 129/Sv and Swiss Webster as main components) (11), CAGGS-CreER (11), and NOD.CB17-Prkdc scid /J (NOD.SCID) mice were purchased from The Jackson Laboratory. p16p19 null mice (B6.129 background) were obtained from the NIH/Mouse Models of Human Cancer Consortium. Mice were bred and maintained at the Joslin Diabetes Center Animal Facility. All animal experiments were approved by the Joslin Diabetes Center Institutional Animal Care and Use Committee.

LUNG METASTASES
Tumor-bearing mice were monitored at least twice weekly for health problems, and were sacrificed once tumors reached a volume of 1 cm 3 or were ill. Lungs were dissected, fixed in 4% paraformaldehyde for 2 h, and embedded in paraffin. Standard www.frontiersin.org H&E stained sections were prepared and evaluated for the presence of metastases by Roderick T. Bronson.

TUMOR TRANSPLANTATION
Tumors were harvested, digested in DMEM + 0.2% collagenase type II (Invitrogen) + 0.05% dispase (Invitrogen) for 90 min at 37°C in a shaking waterbath, triturated to disrupt the remaining tumor pieces, and filtered through a 70 mm cell strainer. Red blood cells were lysed from tumor cell preparations by 3 min incubation in 0.15 M ammonium chloride, 0.01 M potassium bicarbonate solution on ice. Defined numbers of tumor cells were resuspended in 10-15 ml of HBSS with 2% FBS and injected into the gastrocnemius muscles of 1-to 3-month-old, anesthetized NOD.SCID mice using a transdermally inserted dental needle attached to a Hamilton syringe via polyethylene tubing. Recipient muscles were preinjured 24 h before cell implantation by injection of 25 ml of a 0.03 mg/ml solution of cardiotoxin (from Naja mossambica, Sigma) in order to enhance cell engraftment. Mice were screened once weekly for the development of tumors at the injection sites.

Kras; p16p19 null AND SmoM2 MOUSE MYOGENIC TUMORS HAVE DIFFERENT METASTATIC POTENTIAL
The lung is the primary organ affected by distant sarcoma metastases in humans. To assess the metastatic potential of Kras; p16p19 null and SmoM2 tumors, random lung sections obtained from tumor-bearing animals were screened for the presence of metastases. Six of seven mice with Kras; p16p19 null myogenic tumors were found to have lung metastases at the time of death (mice were sacrificed 17-28 days after detection of palpable tumors) (Figure 2). In contrast, 0 of 8 mice with SmoM2 myogenic tumors had lung metastases at the time of death (mice were sacrificed at 38-55 days of age and 5-21 days after detection of palpable tumors). The prevalence of lung metastases in Kras; p16p19 null and SmoM2 myogenic tumor-bearing mice was significantly different (p = 0.001).

Kras; p16p19 null AND SmoM2 MOUSE MYOGENIC TUMORS DIFFER IN TUMOR-REPOPULATING ACTIVITY
Most malignant tumors contain cells that have the capacity to repopulate secondary tumors when transplanted into a susceptible secondary environment, and this assay has been used as a test of the malignancy of distinct tumors and tumor cell subsets (13). To evaluate the tumor-repopulating activity of Kras; p16p19 null and SmoM2 mouse myogenic tumors, viable tumor cells were transplanted into the cardiotoxin-pre-injured gastrocnemius muscles of NOD.SCID mice. The Kras; p16p19 null tumor cell pool contains approximately 70% GFP+ cells and 30% GFP− cells (10). Because tumor-repopulating activity in Kras; p16p19 null tumors resides within the Kras-expressing, GFP+ subset of tumor cells descended from virally infected satellite cells ( Figure S1 in Supplementary Frontiers in Oncology | Pediatric Oncology

DISCUSSION
Our findings highlight differences in the malignant phenotype and behavior of mouse myogenic tumors driven by activation of distinct RMS-relevant oncogenic pathways. Kras; p1619 null myogenic tumors were metastatic to the lungs of the majority of tumorbearing animals and contained high tumor-repopulating activity.
In contrast, SmoM2 tumors did not metastasize and were substantially less effective in repopulating tumors in secondary recipients.
These observations indicate that genetically distinct myogenic tumors in mice display marked differences in their malignant behavior.

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The two model systems described in this study were induced by different experimental methods. SmoM2 tumors originated from Cre-mediated activation of a conditionally expressed transgene. Kras; p16p19 null mouse tumors, on the other hand, were initiated by viral transduction and intramuscular implantation of target satellite cells. We note that Kras; Tp53 −/− mouse myogenic tumors (14,15), induced by Cre-mediated activation of oncogenic hits instead of viral transduction, exhibit a phenotype that closely resembles the Kras; p16p19 null mouse tumors described here. For example, Kras; p16p19 null share their propensity to metastasize to the lungs of tumor-bearing animals with Kras; Tp53 −/− mouse tumors (14). Nevertheless, it is possible that differences in the tumor induction strategy (such as off-target effects of viral transduction) could contribute to the observed differences in malignant behavior between SmoM2 and Kras; p16p19 null mouse myogenic tumors.
Similar to mouse myogenic tumors, human fusion-negative RMS comprises a group of tumors with clear differences in histology, myogenic differentiation state, oncogenic pathway activation, and genetic background. In recent years, subsets of human RMS tumors that exhibit a combination of specific genetic and phenotypic characteristics were distinguished. For example, a subset of human fusion-negative RMS with spindle cell/sclerosing histology was recently found to exhibit diffuse MyoD expression, carry frequent somatic MyoD mutations, and portend a poor prognosis (16,17). Also, children with TP53 germline mutations are predisposed to develop anaplastic RMS at a young age (18), and germline mutations in DICER1 were linked to a genetic susceptibility to develop RMS of the genitourinary tract (19). Future extended (epi-)genotype/phenotype correlations might pinpoint clinically/biologically distinct subgroups of human fusion-negative RMS and identify biomarkers to facilitate prognostication and/or stratification of therapy.

AUTHOR CONTRIBUTIONS
SH, RB, and AW conceived experiments, analyzed data, wrote, and approved of the manuscript.

ACKNOWLEDGMENTS
We thank C. L. Unitt and T. Bowman at the DF/HCC Histopathology Core for help with immunohistochemistry, D. Tchessalova for excellent animal care, and Joyce LaVecchio, Girijesh Burizula, and Atsuya Wakayabashe in the Joslin Diabetes Center Flow Cytometry Core (supported by the Harvard Stem Cell Institute and NIH P30DK036836) for flow cytometry support. This work was funded in part by a Stand Up To Cancer-American Association for Cancer Research Innovative Research Grant (SU2C-AACR-IRG1111; to AW); by grants from the Burroughs-Wellcome Fund and the Harvard Stem Cell Institute (to AW), and by P.A.L.S. Bermuda/St. Baldrick's, ALSF, and Bear Necessities (to SH). Content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other funding agencies.