br Introduction Rhabdomyosarcoma RMS is

Introduction Rhabdomyosarcoma (RMS) is a myogenic tumor that accounts for approximately 50% of all pediatric soft tissue sarcomas. Diagnostic criteria for RMS Thonzonium Bromide on the validation of proteins of the skeletal muscle lineage, such as Desmin, MyoD or Myogenin [1]. RMS cells are persistently kept in a proliferation state and fail to differentiate terminally [2], giving rise to four subtypes named embryonal, alveolar, pleomorphic, and spindle cell/sclerosing, each identified by distinctive genetic, histological and clinical features [3]. The main embryonal and alveolar forms are diagnosed in children under the age of 10 years and in adolescents or young adults, respectively. Embryonal tumors are often characterized by the loss of heterozygosity at chromosome 11p15.5 [4] and activation of the receptor tyrosine kinase/RAS/ERK axis [5], which plays a key role in the tumor growth [[6], [7], [8], [9]], radioresistance [[10], [11], [12]] and metastasis [13]. Alveolar tumors are instead dominated by the t(2;13)(q35;q14) or t(1;13)(q36;q14) chromosomal translocations, which are responsible of the fusion of the paired box 3 and 7 (PAX3 and 7) genes and the 3′ end of the Forkhead box O1 (FOXO1) that generate the chimeric Pax3-FoxO1 or Pax7-FoxO1 oncoproteins, respectively [14]. Despite a multimodal therapy involving chemo- and radiotherapy and surgery can improve the prognosis in most cases, the occurrence of activating RAS mutations, the fusion-positive alveolar histology or the presence of metastases adversely influence the survival rate of RMS patients [15,16]. We previously reported that Caveolin-1 (Cav-1), an ubiquitous protein belonging to a family of three highly conserved members (Cav-1, Cav-2 and Cav-3) [17], can promote tumor growth of embryonal RMS in vitro and in vivo [[18], [19], [20], [21]]. Cav-1 is a scaffolding protein [22] with the ability to increase the biogenesis of caveolae, cholesterol-enriched microdomains of the plasma membrane involved in various cellular processes, such as mechanical stress response [23], endocytosis [24], and signal transduction [25]. The role of Cav-1 in cancer appears to be complex, since its reported ability to modulate diverse cell signaling pathways [26] via the direct binding to a number of receptorial (G proteins, tyrosine-kinase receptors) and non-receptorial proteins (Src, H-Ras, endothelial NOS) mediated by a caveolin scaffolding domain [27]. Indeed, Cav-1 can either behave as a tumor suppressor or oncogene depending on many factors, including the tumor type and stage progression or the presence of post-translational modifications in its primary structure [[28], [29], [30]]. For example, loss of Cav-1 sensitizes to skin tumors in response to carcinogen agents [31], whilst gain of Cav-1 expression is a poor predictor in prostate cancer [32]. Despite this, during advanced stages of cancer metastasis Cav-1 is reported to be often markedly expressed [33], as observed in gastric cancer [34] and melanoma cells [35], as well as to become phosphorylated by members of Src-kinase family [36] to activate pathways linked to cell survival [37]. For example, phosphorylated Cav-1 has been reported to influence focal adhesion dynamics through Src kinase and Rho GTPases, therefore enhancing cell polarization, directional migration and invasion in metastatic cancer cells [[38], [39], [40], [41], [42]]. As a result, the occurrence of phosphorylated Cav-1 is thought to predict unfavorable outcome by correlating with anchorage-independent cell growth, migration, invasiveness and multidrug resistance. In this work, by using a gain of function approach we demonstrated through an experimental in vivo metastasis assay that Cav-1 facilitates the dissemination of the embryonal RD tumor cells through cooperation with Erk signaling.
Materials and methods
Results
Discussion Metastatic dissemination is the leading cause of death in cancer patients. For RMS patients long-term survival for metastatic tumors remains low, being <20% for alveolar and 60% for embryonal subtypes [44]. The identification of molecular targets eliciting metastasis dissemination is therefore a challenge to improve current RMS therapeutic regimens. Over the past years we have demonstrated that Cav-1 is preferentially expressed in embryonal RMS tumors featured by a poor degree of myogenic differentiation [18]. In addition, we found that its increased or decreased expression in human engineered embryonal RD cells facilitates or dampens tumor growth in vitro and in vivo, respectively [20,21]. Here, by means of an experimental metastasis assay, we found that the increased Cav-1 levels can also facilitate the metastatic dissemination of embryonal RMS. Metastasis process involves four major steps: detachment of cancer cells from their primary loci, their entry into circulation (intravasation), their exit from circulation (extravasation), and survival and growth in a distant organ site [45]. In this regard, it is important to underline that our experimental model based on the intravenous injection of cancer cells provides a means to evaluate organ colonization by cancer cells rather than the overall metastatic process [46]. However, the aggressive metastatic lines we generated are useful tools for identifying genes or molecular pathways related to metastatic progression of embryonal RMS.