The decreased metastatic potential of rhabdomyosarcoma cells obtained through MET receptor downregulation and the induction of differentiation.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. The MET receptor has an important role in the biology of RMS, and its overexpression and hyperactivation correlate with the metastatic ability of RMS. Consequently, interfering with MET expression or functionality may constitute a sound strategy for reducing the progression and metastatic potential of RMS. Our study reveals that downregulation of the MET receptor leads to changes in the morphology of ARMS cell in vivo. Tumors acquire a spindle shape that is characteristic of muscle fibers. Inhibition of MET expression or function leads to (i) a decreased expression of the early myogenic marker MyoD, (ii) a decreased ability of ARMS cells to metastasize to bone marrow cavities, (iii) downregulation of CXCR4 receptor expression and (iv) a decreased migration of MET-depleted cells towards gradients of HGF and SDF-1. Finally, we demonstrate that in vitro differentiation of alveolar RMS cells decreases their metastatic behavior by reducing both the expression of the MET and CXCR4 receptors and their migratory response to HGF and SDF-1. These findings suggest that blockers of MET receptor function and inducers of RMS cells differentiation may be clinically useful for reducing the aggressiveness and metastatic potential of RMS and may have significant implications for its treatment.

High anti tumor activity against rhabdomyosarcoma cells and low normal cells cytotoxicity of heat shock protein 90 inhibitors, with special emphasis on 17-[2-(pyrrolidin-1-yl)ethyl]-aminno-17-demethoxygeldanamycin.

Rhabdomyosarcoma is a highly metastatic tumor, mostly observed in children and adolescence. When diagnosed at early stages it is mostly curable. However, in advanced or metastatic stages the 5-years survival rate is below 20%. Thus, new treatment strategies for this tumor are needed. In this paper we showed that HSP90 inhibitors, geldanamycin and its analogs, can profoundly affect proliferation of rhabdomyosarcoma cells. We also showed that blocking of HSP90 function induces apoptosis of tumor cells and downregulates expression of anti apoptotic protein AKT. Cells exposed to geldanamycin and its analogs exhibit strong reduction of MET receptor expression and subsequent inhibition of HGF-dependent tumor cells migration and invasion. Interestingly, at concentrations sufficient to block tumor cells growth and motility, the 17AEP-GA, 17AAG and 17DMAP-GA were not toxic or only slightly toxic toward normal hematopoietic, mesenchymal and endothelial cells. This could be due to low HSP90 expression both at mRNA and protein level in these cells. Collectively, our findings suggest that blocking HSP90 action through geldanmycins could be in the future a part of new therapeutic strategies in rhabdomyosarcoma treatment.

Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.

Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 microg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.

Homotypic cell-cell contacts stimulate the motile activity of rat prostate cancer cells.

OBJECTIVE: To characterize the effect of homotypic cell-to-cell collisions upon the motile activities of two rat prostatic cancer cell lines of markedly different metastatic potential. MATERIALS AND METHODS: The movements of strongly and weakly metastatic MAT-LyLu and AT-2 cells, respectively, were recorded under an inverted microscope at 37 degrees C. The motile activities of the cells at various cell densities were characterized quantitatively by computer-aided tracking methods and image analysis. The following variables were assessed: speed of movement, final displacement, coefficient of movement efficiency, diffusion constant and positive flow. RESULTS: MAT-LyLu and AT-2 cells showed only limited motile activity in sparse cultures where there was little contact amongst the cells. However, under these and all other subsequent conditions tested, the motile activity of the MAT-LyLu cells was higher than the AT-2 cells. As the density of the cultured cells was increased (leading to more cell-to-cell contacts) there was a significant increase in motility. This effect was more pronounced for the AT-2 than for the MAT-LyLu cells, resulting in visible acceleration of movement by direct physical contact among the colliding cells. The motile activities of the tumour cells was only slightly affected by conditioned media. CONCLUSION: Homotypic collisions between migrating prostatic cancer cells can strongly stimulate their motility. The effect of increased contact is greater on the weakly metastatic cells, such that at high cell density, the difference in the motilities of weak and strong metastatic cells is greatly reduced.