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.

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 (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1 À/À ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1 À/À mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1 À/À animals have reduced quiescence and regenerative potential. Young HO-1 À/À HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1 +/+ HSCs transplanted into HO-1 À/À recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1 À/À HSCs to the HO-1 +/+ recipients recovers the regenerative potential of HO-1 À/À HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.EMBO Reports (2020) 21: e47895 Until now, the mechanisms found to contribute to the aging of HSCs have been mostly intrinsic to the HSCs [6,15]. These include age-related accumulation of mutations and cell-autonomous changes in the transcriptome and epigenome of HSCs [6,7,[16][17][18][19].Although HSC-extrinsic factors from the local bone marrow (BM) environment of HSCs-the HSC niche-or systemic factors are critical for HSC maintenance [20][21][22], little is known about their contribution to HSC aging.Recent findings indicate that HSCs occupy a perivascular niche and localize in direct proximity to endothelial cells (ECs) and mesenchymal stromal cells (MSCs) surrounding vessels [23,24]. Among the many cell types in the HSC niche, the ECs and MSCs constitute the main source of stromal cell-derived factor 1a (SDF-1a) and stem cell factor (SCF)-extrinsic factors critical for HSC maintenance [25][26][27][28]. Specific deletion of either Sdf1a or Scf in either ECs or MSCs causes hematopoietic collapse or triggers overactivation of HSCs and their release from the niche [22,[25][26][27]. J endomucin CD31 HO-1 Figure 1. ª 2019 The Authors EMBO reports 21: e47895 | 2020 ****P < 0.0001. Data are shown as mean AE SEM. IECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean AE SEM. *P < 0.05, ***P < 0.001, two-tailed unpaired t-test. JMiddle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean AE SEM. **P < 0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1 À/À bone marrow section is provided in Appendix Fig S1. 4 of 21 EMBO reports 21: e47895 | 2020 ª 2019 The Authors H Only 1 out of 267 DEGs identified in non-tran...

Both MSC Hmox1 and Hmox1 showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1 and Hmox1 cells showed similar survival in response to 50 μmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1 bone marrow-derived proangiogenic cells (BDMC). Hmox1 MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 μmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1 treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1 do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1 show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.