The synthetic retinoid ST1926 as a novel therapeutic agent in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, only moderate improvements in survival have been made for patients with advanced disease. Retinoic acid is a differentiation agent that has shown some antitumor efficacy in RMS cells in vitro; however, the effects are of low magnitude. E-3-(4'-hydroxyl-3'-adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel orally available synthetic atypical retinoid, shown to have more potent activity than retinoic acid in several types of cancer cells. We used in vitro and in vivo models of RMS to explore the efficacy of ST1926 as a possible therapeutic agent in this sarcoma. We found that ST1926 reduced RMS cell viability in all tested alveolar (ARMS) and embryonal (ERMS) RMS cell lines, at readily achievable micromolar concentrations in mice. ST1926 induced an early DNA damage response (DDR), which led to increase in apoptosis, in addition to S-phase cell cycle arrest and a reduction in protein levels of the cell cycle kinase CDK1. Effects were irrespective of TP53 mutational status. Interestingly, in ARMS cells, ST1926 treatment decreased PAX3-FOXO1 fusion oncoprotein levels, and this suppression occurred at a post-transcriptional level. In vivo, ST1926 was effective in inhibiting growth of ARMS and ERMS xenografts, and induced a prominent DDR. We conclude that ST1926 has preclinical efficacy against RMS, and should be further developed in this disease in clinical trials.

Exosome-Mediated Paracrine Signaling Unveils miR-1246 as a Driver of Aggressiveness in Fusion-Negative Rhabdomyosarcoma.

Rhabdomyosarcoma is a pediatric cancer associated with aggressiveness and a tendency to develop metastases. Fusion-negative rhabdomyosarcoma (FN-RMS) is the most commonly occurring subtype of RMS, where metastatic disease can hinder treatment success and decrease survival rates. RMS-derived exosomes were previously demonstrated to be enriched with miRNAs, including miR-1246, possibly contributing to disease aggressiveness. We aimed to decipher the functional impact of exosomal miR-1246 on recipient cells and its role in promoting aggressiveness. Treatment of normal fibroblasts with FN-RMS-derived exosomes resulted in a significant uptake of miR-1246 paired with an increase in cell proliferation, migration, and invasion. In turn, delivery of miR-1246-mimic lipoplexes promoted fibroblast proliferation, migration, and invasion in a similar manner. Conversely, when silencing miR-1246 in FN-RMS cells, the resulting derived exosomes demonstrated reversed effects on recipient cells' phenotype. Delivery of exosomal miR-1246 targets GSK3ß and promotes ß-catenin nuclear accumulation, suggesting a deregulation of the Wnt pathway, known to be important in tumor progression. Finally, a pilot clinical study highlighted, for the first time, the presence of high exosomal miR-1246 levels in RMS patients' sera. Altogether, our results demonstrate that exosomal miR-1246 has the potential to alter the tumor microenvironment of FN-RMS cells, suggesting its potential role in promoting oncogenesis.

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.

Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts.

Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes.

p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells.

The restoration of p53 has been suggested as a therapeutic approach in tumors. However, the timing of p53 restoration in relation to its efficacy during tumor progression still is unclear. We now show that the restoration of p53 in murine premalignant proliferating pineal lesions resulted in cellular senescence, while p53 restoration in invasive pineal tumors did not. The effectiveness of p53 restoration was not dependent on p19(Arf) expression but showed an inverse correlation with Mdm2 expression. In tumor cells, p53 restoration became effective when paired with either DNA-damaging therapy or with nutlin, an inhibitor of p53-Mdm2 interaction. Interestingly, the inactivation of p53 after senescence resulted in reentry into the cell cycle and rapid tumor progression. The evaluation of a panel of human supratentorial primitive neuroectodermal tumors (sPNET) showed low activity of the p53 pathway. Together, these data suggest that the restoration of the p53 pathway has different effects in premalignant versus invasive pineal tumors, and that p53 activation needs to be continually sustained, as reversion from senescence occurs rapidly with aggressive tumor growth when p53 is lost again. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene is intact but the pathway is inactive in the majority of examined tumors.

CDK2 transcriptional repression is an essential effector in p53-dependent cellular senescence-implications for therapeutic intervention.

UNLABELLED: Cellular senescence, a form of cell-cycle arrest, is a tumor-suppressor mechanism triggered by multiple tumor-promoting insults, including oncogenic stress and DNA damage. The role of cyclin-dependent kinase 2 (CDK2) regulation has been evaluated in models of replicative senescence, but little is known regarding its role in other senescence settings. Using in vitro and in vivo models of DNA damage-and oncogene-induced cellular senescence, it was determined that activation of the tumor-suppressor protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was sufficient to bypass p53-dependent senescence, and CDK2-specific inhibition, either pharmacologically (CVT313) or by use of a dominant-negative CDK2, was sufficient to induce early senescence. Pharmacologic inhibition of CDK2 in an in vivo model of pineal tumor decreased proliferation and promoted early senescence, and it also decreased tumor penetrance and prolonged time to tumor formation in animals lacking p53. In conclusion, for both oncogene- and DNA damage-induced cellular senescence, CDK2 transcript and protein are decreased in a p53- and RB-dependent manner, and this repression is necessary for cell-cycle exit during senescence. IMPLICATIONS: These data show that CDK2 inhibition may be useful for cancer prevention in premalignant hyperproliferative lesions, as well as established tumors.