Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncogene. Despite its discovery nearly two decades ago, the mechanisms by which PAX3-FOXO1 drives tumor development are not well characterized. Previously, we reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence checkpoints. We have now found that this bypass occurs in part through PAX3-FOXO1–mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member. RASSF4 expression was upregulated in PAX3-FOXO1–positive aRMS cell lines and tumors. Enhanced RASSF4 expression promoted cell cycle progression, senescence evasion, and tumorigenesis through inhibition of the Hippo pathway tumor suppressor MST1. We also found that the downstream Hippo pathway target Yes-associated protein 1 (YAP), which is ordinarily restrained by Hippo signaling, was upregulated in RMS tumors. These data suggest that Hippo pathway dysfunction promotes RMS. This work provides evidence for Hippo pathway suppression in aRMS and demonstrates a progrowth role for RASSF4. Additionally, we identify a mechanism used by PAX3-FOXO1 to inhibit MST1 signaling and promote tumorigenesis in aRMS.

Purpose: Rhabdomyosarcoma (RMS) is a malignancy with features of skeletal muscle, and the most common soft tissue sarcoma of childhood. Survival for high-risk groups is approximately 30% at 5 years and there are no durable therapies tailored to its genetic aberrations. During genetic modeling of the common RMS variants, embryonal RMS (eRMS) and alveolar RMS (aRMS), we noted that the receptor tyrosine kinase (RTK) fibroblast growth factor receptor 4 (FGFR4) was upregulated as an early event in aRMS. Herein, we evaluated the expression of FGFR4 in eRMS compared with aRMS, and whether FGFR4 had similar or distinct roles in their tumorigenesis. Experimental Design: Human RMS cell lines and tumor tissue were analyzed for FGFR4 expression by immunoblot and immunohistochemistry. Genetic and pharmacologic loss-of-function of FGFR4 using virally transduced short hairpin RNA (shRNA) and the FGFR small-molecule inhibitor PD173074, respectively, were used to study the role of FGFR4 in RMS cell lines in vitro and xenografts in vivo. Expression of the antiapoptotic protein BCL2L1 was also examined. Results: FGFR4 is expressed in both RMS subtypes, but protein expression is higher in aRMS. The signature aRMS gene fusion product, PAX3-FOXO1, induced FGFR4 expression in primary human myoblasts. In eRMS, FGFR4 loss-of-function reduced cell proliferation in vitro and xenograft formation in vivo. In aRMS, it diminished cell survival in vitro. In myoblasts and aRMS, FGFR4 was necessary and sufficient for expression of BCL2L1 whereas in eRMS, this induction was not observed, suggesting differential FGFR4 signaling. Conclusion: These studies define dichotomous roles for FGFR4 in RMS subtypes, and support further study of FGFR4 as a therapeutic target. Clin Cancer Res; 18(14); 3780–90. ©2012 AACR.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including loss-of-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16^INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16^INK4A/p14^ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16^INK4A/p14^ARF. These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state. [Cancer Res 2008;68(23):9583–8]

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of <30%. ARMS is characterized by a chromosomal translocation generating the PAX3-FKHR fusion gene. However, ectopic expression of PAX3-FKHR often induces inhibition of cell proliferation, or cell death, when expressed in nonmuscle cells. This prompted us to explore the effect of expressing PAX3-FKHR in more relevant cells, specifically primary human skeletal muscle cells because these cells can be converted to a tumorigenic state that mimics rhabdomyosarcoma. PAX3-FKHR expression promoted both fetal and postnatal primary human skeletal muscle cell precursors to bypass the senescence growth arrest checkpoint. This bypass was accompanied by epigenetic DNA methylation of the p16INK4A promoter and correspondingly a loss of expression of this tumor suppressor. Knockdown of p16INK4A cooperated with PAX3-FKHR to drive proliferation past senescence, whereas reintroduction of wild-type p16INK4A in post-senescent cells caused growth arrest. Thus, PAX3-FKHR acts in concert with loss of p16INK4A to promote inappropriate proliferation of skeletal muscle cells. This association between PAX3-FKHR expression and p16INK4A loss was seen in human ARMS tumor tissue, as both human rhabdomyosarcoma cell lines and tissue microarrays showed a trend toward down-regulation of p16INK4A protein in alveolar subsets. We surmise that the generation of the PAX3-FKHR fusion protein may require loss of p16INK4A to promote malignant proliferation of skeletal muscle cells as an early step in ARMS tumorigenesis. [Cancer Res 2007;67(14):6691–9]