Caveolin-1 enhances metastasis formation in a human model of embryonal rhabdomyosarcoma through Erk signaling cooperation.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue tumor classified in two major subtypes namely embryonal and alveolar, which have distinctive histopathological and genetic signatures and worse outcomes in the presence of metastases. Here, in order to evaluate the role of Caveolin-1 (Cav-1) in embryonal RMS dissemination, we employed an experimental in vivo metastasis assay using immunodeficient NOD/SCID mice. We found that the intravenous injection of human RD cells engineered for Cav-1 overexpression promoted the formation of lung metastases compared to parental cells. The arisen metastases were isolated and cultured in vitro to establish two derivative lines that showed greater metastatic capacity, as detected by performing in vivo metastasis and tumor spheroid invasion assays. Compared to parental cells, all metastatic lines were characterized by an increase in cell proliferation, migration and invasiveness that were downregulated by synthetic inhibition of Erk pathway. The metastatic cells showed a marked cell apoptosis induced by nutrient deprivation and consistent loss of differentiation characterized by depletion of MyoD and Myogenin factors. Furthermore, they showed marked changes in cell size, a re-organization of the three-dimensional cytoskeleton characterized by an increased actin stress fiber content, and increased adhesion and angiogenic properties. Collectively, these data provide new insights into Cav-1-driven metastatic process of embryonal RMS through cooperation of the Erk signaling pathway. Furthermore, our derivative metastatic lines represent useful tools for identifying genes or molecular pathways that regulate the metastatic progression of embryonal RMS.

Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma.

Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines.

Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.

Phosphocaveolin-1 enforces tumor growth and chemoresistance in rhabdomyosarcoma.

Caveolin-1 (Cav-1) can ambiguously behave as either tumor suppressor or oncogene depending on its phosphorylation state and the type of cancer. In this study we show that Cav-1 was phosphorylated on tyrosine 14 (pCav-1) by Src-kinase family members in various human cell lines and primary mouse cultures of rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma affecting childhood. Cav-1 overexpression in the human embryonal RD or alveolar RH30 cells yielded increased pCav-1 levels and reinforced the phosphorylation state of either ERK or AKT kinase, respectively, in turn enhancing in vitro cell proliferation, migration, invasiveness and chemoresistance. In contrast, reducing the pCav-1 levels by administration of a Src-kinase inhibitor or through targeted Cav-1 silencing counteracted the malignant in vitro phenotype of RMS cells. Consistent with these results, xenotransplantation of Cav-1 overexpressing RD cells into nude mice resulted in substantial tumor growth in comparison to control cells. Taken together, these data point to pCav-1 as an important and therapeutically valuable target for overcoming the progression and multidrug resistance of RMS.

Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes.

Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt-chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl2 doses (from 5 to 200µM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl2 doses for prolonged time points. Furthermore, CoCl2 treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical `pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy.

Implications for the mammalian sialidases in the physiopathology of skeletal muscle.

The family of mammalian sialidases is composed of four distinct versatile enzymes that remove negatively charged terminal sialic acid residues from gangliosides and glycoproteins in different subcellular areas and organelles, including lysosomes, cytosol, plasma membrane and mitochondria. In this review we summarize the growing body of data describing the important role of sialidases in skeletal muscle, a complex apparatus involved in numerous key functions and whose functional integrity can be affected by various conditions, such as aging, chronic diseases, cancer and neuromuscular disorders. In addition to supporting the proper catabolism of glycoconjugates, sialidases can affect different signaling pathways by desialylation of many receptors and modulation of ganglioside content in cell membranes, thus actively participating in myoblast proliferation, differentiation and hypertrophy, insulin responsiveness and skeletal muscle architecture.

Rhabdomyosarcomas: an overview on the experimental animal models.

Rhabdomyosarcomas (RMS) are aggressive childhood soft-tissue malignancies deriving from mesenchymal progenitors that are committed to muscle-specific lineages. Despite the histopathological signatures associated with three main histological variants, termed embryonal, alveolar and pleomorphic, a plethora of genetic and molecular changes are recognized in RMS. Over the years, exposure to carcinogens or ionizing radiations and gene-targeting approaches in vivo have greatly contributed to disclose some of the mechanisms underlying RMS onset. In this review, we describe the principal distinct features associated with RMS variants and focus on the current available experimental animal models to point out the molecular determinants cooperating with RMS development and progression.

Point mutated caveolin-3 form (P104L) impairs myoblast differentiation via Akt and p38 signalling reduction, leading to an immature cell signature.

Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-ß-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-ß signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.