Signaling pathways in Rhabdomyosarcoma invasion and metastasis.

Rhabdomyosarcoma (RMS) is an aggressive childhood mesenchymal tumor with two major molecular and histopathologic subtypes: fusion-positive (FP)RMS, characterized by the PAX3-FOXO1 fusion protein and largely of alveolar histology, and fusion-negative (FN)RMS, the majority of which exhibit embryonal tumor histology. Metastatic disease continues to be associated with poor overall survival despite intensive treatment strategies. Studies on RMS biology have provided some insight into autocrine as well as paracrine signaling pathways that contribute to invasion and metastatic propensity. Such pathways include those driven by the PAX3-FOXO1 fusion oncoprotein in FPRMS and signaling pathways such as IGF/RAS/MEK/ERK, PI3K/AKT/mTOR, cMET, FGFR4, and PDGFR in both FP and FNRMS. In addition, specific cytoskeletal proteins, G protein coupled receptors, Hedgehog, Notch, Wnt, Hippo, and p53 pathways play a role, as do specific microRNA. Paracrine factors, including secreted proteins and RMS-derived exosomes that carry cargo of protein and miRNA, have also recently emerged as potentially important players in RMS biology. This review summarizes the known factors contributing to RMS invasion and metastasis and their implications on identifying targets for treatment and a better understanding of metastatic RMS.

Proteomic Profiling of Rhabdomyosarcoma-Derived Exosomes Yield Insights into Their Functional Role in Paracrine Signaling.

Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They have been demonstrated to contribute to the metastatic progression of tumor cells through paracrine signaling. Tumor exosomes contain intact and functional proteins, mRNA and miRNA that may alter the cellular environment to favor tumor growth. We evaluated the protein cargo of exosomes derived from the childhood tumor rhabdomyosarcoma (RMS) and the molecular pathways they are implicated in to decipher their role in the progression of this aggressive disease. We conducted a mass spectrometry analysis of exosome content isolated from five RMS cell lines: three of embryonal RMS (ERMS) and two of alveolar RMS (ARMS) histology and verified results by multiple reaction monitoring and western blot analyses. Results revealed 161 common proteins in ERMS-derived exosomes and 122 common proteins in ARMS-derived exosomes, of which 81 proteins were common to both subtypes. Using both PANTHER gene classification and Pathway Studio software, we assessed the perturbed biological processes and altered pathways in which the exosomal proteins are involved. The 81 commonly expressed proteins included those involved in "cell-signaling," "cell-movement," and "cancer." Pathways engaging the identified proteins revealed 37 common pathways including "integrin signaling pathway," "inflammation mediated by chemokine and cytokine signaling pathway," and "angiogenesis." Finally, a comparison of exosomal proteins of RMS cells with publicly available datasets from other cancer cells revealed that 36 proteins are specific and endogenous to the RMS-exosomes. Taken together, our results reveal that RMS-derived exosomes carry a protein cargo that contributes to conserved cellular signaling networks across multiple cell lines, and we also identify RMS exosome-specific proteins that should be further evaluated as possible novel biomarkers for this tumor.

CD147 Promotes Tumorigenesis via Exosome-Mediated Signaling in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive childhood soft-tissue tumor, with propensity for local invasion and distant metastasis. Exosomes are secreted vesicles that mediate paracrine signaling by delivering functional proteins and miRNA to recipient cells. The transmembrane protein CD147, also known as Basigin or EMMPRIN, is enriched in various tumor cells, as well as in tumor-derived exosomes, and has been correlated with poor prognosis in several types of cancer, but has not been previously investigated in RMS. We investigated the effects of CD147 on RMS cell biology and paracrine signaling, specifically its contribution to invasion and metastatic phenotype. CD147 downregulation diminishes RMS cell invasion and inhibits anchorage-independent growth in vitro. While treatment of normal fibroblasts with RMS-derived exosomes results in a significant increase in proliferation, migration, and invasion, these effects are reversed when using exosomes from CD147-downregulated RMS cells. In human RMS tissue, CD147 was expressed exclusively in metastatic tumors. Altogether, our results demonstrate that CD147 contributes to RMS tumor cell aggressiveness, and is involved in modulating the microenvironment through RMS-secreted exosomes. Targeted inhibition of CD147 reduces its expression levels within the isolated exosomes and reduces the capacity of these exosomes to enhance cellular invasive properties.

Effects of the Oncoprotein PAX3-FOXO1 on Modulation of Exosomes Function and Protein Content: Implications on Oxidative Stress Protection and Enhanced Plasticity.

Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma classified into two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS). ARMS subtype is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 (P3F) that drives oncogenic cellular properties. To understand the role of the fusion oncoprotein in paracrine signaling, we focused on secreted exosomes, which have been demonstrated to contribute to metastasis in multiple tumor types. Advanced Proteomics-bioinformatics analysis of the protein cargo of exosomes isolated from C2C12 myoblasts transduced with P3F fusion gene revealed 52 deregulated proteins compared to control cells, with 26 enriched and 26 depleted proteins. Using both PANTHER gene classification and Ingenuity Pathway Analysis (IPA) software, we found that the main biological processes in which the 52 deregulated proteins are involved, include "catalytic activity," "binding," "metabolic process," and "cellular process." The pathways engaging the 26 enriched proteins include the "14-3-3 mediated signaling," "cell cycle," and "ERK5, VEGF, IGF1,and p70S6K signaling." Furthermore, the main nodes in which deregulated exosome proteins and miRNAs intersected revealed pathways conferring protection from stress and promoting plasticity. Based on the bioinformatics analysis and the altered exosome proteome profile, we performed biochemical functional analysis to study the diverse properties of these exosomes where angiogenesis, stemness, and anti-oxidative stress properties were validated using different platforms. P3F-modulated exosomes activated ERK, 4-EBP1, and MMP-2 in recipient cells, and enhanced angiogenesis and stemness. In addition, P3F led to lower cellular reactive oxygen species levels and enhanced resistance against oxidative stress; and treatment of stromal cells with P3F-modulated exosomes also conferred protection against exogenous oxidative stress. Our findings highlight the role of P3F fusion protein in modulating exosome cargo to confer a protective effect on recipient cells against oxidative stress and to promote plasticity and survival, potentially contributing to the known aggressive phenotype of the fusion gene-positive subtype of RMS.

The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by exosomal miR-486.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker.

The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.