Rhabdomyosarcoma (RMS) is a mesenchymal tumor of soft tissue in children that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS (ARMS), characterized by a low myogenic differentiation status and high aggressiveness. In RMS patients SNAIL level increases with higher stage. Moreover, SNAIL level negatively correlates with MYF5 expression. The differentiation of human ARMS cells diminishes SNAIL level. SNAIL silencing in ARMS cells inhibits proliferation and induces differentiation in vitro, and thereby completely abolishes the growth of human ARMS xenotransplants in vivo. SNAIL silencing induces myogenic differentiation by upregulation of myogenic factors and muscle-specific microRNAs, such as miR-206. SNAIL binds to the MYF5 promoter suppressing its expression. SNAIL displaces MYOD from E-box sequences (CANNTG) that are associated with genes expressed during differentiation and G/C rich in their central dinucleotides. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting ARMS cell myogenic differentiation. In differentiating ARMS cells SNAIL forms repressive complex with histone deacetylates 1 and 2 (HDAC1/2) and regulates their expression. Accordingly, in human myoblasts SNAIL silencing induces differentiation by upregulation of myogenic factors. Our data clearly point to SNAIL as a key regulator of myogenic differentiation and a new promising target for future ARMS therapies.

Abstract:The efficacy of cancer treatments is often limited and associated with substantial toxicity.

Rhabdomyosarcoma (RMS) is a predominant soft tissue tumor in children and adolescents. For high-grade RMS with metastatic involvement, the 3-year overall survival rate is only 25 to 30%. Thus, understanding the regulatory mechanisms involved in promoting the metastasis of RMS is important. Here, we demonstrate for the first time that the SNAIL transcription factor regulates the metastatic behavior of RMS both in vitro and in vivo. SNAIL upregulates the protein expression of EZRIN and AKT, known to promote metastatic behavior, by direct interaction with their promoters. Our data suggest that SNAIL promotes RMS cell motility, invasion and chemotaxis towards the prometastatic factors: HGF and SDF-1 by regulating RHO, AKT and GSK3b activity. In addition, miRNA transcriptome analysis revealed that SNAIL-miRNA axis regulates processes associated with actin cytoskeleton reorganization. Our data show a novel role of SNAIL in regulating RMS cell metastasis that may also be important in other mesenchymal tumor types and clearly suggests SNAIL as a promising new target for future RMS therapies.

Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and young adulthood. Conventional treatment consisting of surgery, chemotherapy and radiotherapy can be insufficient, as long-term survival chances decrease dramatically when cancer recurrence occurs. Due to this fact, efficient treatment of this cancer is still a demanding issue, thus, novel and innovative therapies have to be considered as a part of combined treatment. In the present study, we present effective suicide gene therapy of rhabdomyosarcoma cell line Rh30 involving herpes simplex thymidine kinase (HSV-TK) and ganciclovir (GCV). Transduction of rhabdomyosarcoma cells using lentiviral vectors allowed efficient introduction of HSV-TK gene. In this study we proved high susceptibility of modified cells to ganciclovir resulting in eradication of cancer cells both in vitro and in vivo. Our data revealed strong gap junctional intercellular communication in examined cell line responsible for elimination of unmodified cells by bystander effect, even if HSV-TK-expressing cells comprise only 20% of cultured cells. Moreover, investigated approach is also efficient in vivo, where complete remission of tumors upon only 14 days of systemic administration of GCV can be observed. Obtained results suggest that HSV-TK suicide gene therapy is very promising concept in future clinical studies concerning rhabdomyosarcoma.

Abstract:Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

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The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

The present article demonstrates selective cytotoxicity against cancer cells of the complexes [Co(LD)2]I2∙CH3OH (1), [CoLD(NCS)2] (2) and [VOLD(NCS)2]∙C6H5CH3 (3) containing the dipodal tridentate ligand LD = N,N-bis(3,5-dimethylpyrazol-1-ylmethyl)amine), formed in situ. All tested complexes expressed greater anticancer activities and were less toxic towards noncancerous cells than cisplatin. Cobalt complexes (1 and 2) combined high cytotoxicity with selectivity towards cancer cells and caused massive tumour cell death. The vanadium complex (3) induced apoptosis specifically in cancer cells and targeted proteins, controlling their invasive and metastatic properties. The presented experimental data and computational prediction of drug ability of coordination compounds may be helpful for designing novel and less toxic metal-based anticancer species with high specificities towards tumour cells.