The mechanisms by which Cav enhances embryonal
The mechanisms by which Cav-1 enhances embryonal tumor progression are currently unknown. Given the documented ability of Cav-1 to drive increased biogenesis of caveolae and accelerate the flow of nutrients (mostly glucose and fatty acids) across the membrane, as observed in particular in adipocytes [47,48], we cannot rule out that a similar mechanism may favor the rapid expansion of metastatic RMS lines. According to this, after nutrient deprivation the metastatic lines were characterized by a marked cell death compared to parental cells, a hallmark commonly detected in flecainide acetate australia featured by high metabolic demands, such as tumor cells indeed . Our data convincingly suggest a critical role for Cav-1 in facilitating the Erk signaling, even if the precise mechanisms through which this occurs remain to be addressed. Indeed, although Cav-1 has first been described in epithelial CHO cells to act as a negative regulator of Erk pathway by direct binding to Erk1/2 proteins , in other cell models it was reported to act as a positive regulator, as observed in colon cancer cells  or in metastatic Ewing sarcoma lines . Elevated Erk signaling is a master driver of embryonal  and pleomorphic RMS  formation. Among the multitude of functions, the Erk pathway also plays a prominent role in overwhelming the differentiation capacity of RMS cells through mechanisms that mediate MyoD inactivation [54,55] and repression of Myogenin expression . Consistent with this, the established metastatic RDF0, RDF1 and RDF2 lines were characterized by low expression of MyoD (already during cell proliferation) and Myogenin, and loss of differentiation capacity. These data therefore suggest that the cooperation between Cav-1 and the Erk signaling may support increased tumorigenic properties through the maintenance of a dedifferentiated state. Interestingly, CAV-1 was indicated as one of the five genes (BMP4, MYF5, DLK1, PAX7, CAV-1) that predict the occurrence of undifferentiated pleomorphic sarcoma (UPS) , which are more aggressive variants than embryonal RMS, lacking consistent histological, immunohistochemical, ultrastructural, or molecular markers . Dedifferentiation is a hallmark found in metastases that are commonly enriched in cancer stem cells (CSCs), the cell subpopulation having the intrinsic ability to self-renew and propagate the entire intratumor heterogeneity . Commonly, as long as tumor progresses, differentiation markers are lost and stem/progenitor markers become predominantly expressed due to cell enrichment in CSCs. To establish whether Cav-1 expression through Erk cooperation could dictate a dedifferentiation program in embryonal RMS, it will be important to characterize whether the enrichment in stem/progenitor markers could occur in three-dimensional RDF1 and RDF2 rhabdospheroids. Our data also indicate that Cav-1 through Erk signaling positively influences migration and invasiveness of metastatic RD lines. However, it is important to underline that the different metastatic lines, though exhibiting increasing metastatic properties (RDF2 and RDF1 lines were more invasive than RDF0 as determined in vivo and by tumor spheroid assay), showed similar levels in the Erk pathway activation, indicating that additional pathways contribute to enhanced metastatic potential. Consistent with this, the synthetic Erk inhibition in the metastatic lines was not sufficient to fully abrogate the migratory and invasive properties. In this regard, we found Cav-1 to be tyrosine-phosphorylated, and this mechanism known to be dependent on Src-kinase family members  has been recently proposed to redistribute the protein in the cytosolic compartments in order to facilitate the interaction with intracellular partners . Since we actually found Cav-1 mainly localizing in intracellular compartments of RD cells away from the plasma membrane, it is reasonable to think that the intracellular accumulation of pCav-1 might be responsible for the modulation of pro-migratory and pro-invasive pathways. In this context, pCav-1 has been documented to enhance migration and invasion in metastatic cancer cells by influencing focal adhesions [, , , , ]. These large, dynamic protein complexes are made up by several proteins, including integrins, vinculin, talin, α-actinin, and focal adhesion kinase (FAK), through which intracellular actin bundles of a cell connects to the extracellular matrix during cell adhesion, morphogenesis and mechanotransduction processes . In view of the changes in cell size and shape observed in RMS metastatic lines, we cannot rule out the possibility that pCav-1 may influence the stress fibers dynamics to increase the migratory and invasive properties. To date, FAK inhibition was reported to decrease tumorigenic properties in different embryonal and alveolar RMS cell lines . Hence, the availability of highly metastatic RMS lines provides a unique tool to validate the potential relationship between pCav-1 and focal adhesions over the dissemination process.