Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.

The modulation of iron metabolism affects the Rhabdomyosarcoma tumor growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is an aggressive rare neoplasm that derives from mesenchymal cells, which frequently develops resistance to the current therapies and the formation of metastases. Thus, new therapies are needed. The alteration of iron metabolism in cancer cells was effective in reducing the progression of many tumors but not yet investigated in RMS. Here we investigated the effect of iron modulation in RMS both in vitro and in vivo. We first characterized the most used RMS cell lines representing the most common subtypes, embryonal (ERMS, RD cells) and alveolar (ARMS, RH30 cells), for their iron metabolism, in basal condition and in response to its modulation. Then we investigated the effects of both iron overload and chelation strategies in vitro and in vivo. RMS cell lines expressed iron-related proteins, even if at lower levels compared to hepatic cell lines and they are correctly modulated in response to iron increase and deprivation. Interestingly, the treatment with different doses of ferric ammonium citrate (FAC, as iron source) and with deferiprone (DFP, as iron chelator), significantly affected the cell viability of RD and RH30. Moreover, iron supplementation (in the form of iron dextran) or iron chelation (in the form of DFP) were also effective in vivo in inhibiting the tumor mass growth both derived from RD and RH30 with iron chelation treatment the most effective one. All the data suggest that the iron modulation could be a promising approach to overcome the RMS tumor growth. The mechanism of action seems to involve the apoptotic cell death for both iron supplementation and chelation with the concomitant induction of ferroptosis in the case of iron supplementation.

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

Translational Implications for Radiosensitizing Strategies in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence that includes FP-RMS, harboring the fusion oncoprotein PAX3/7-FOXO1 and FN-RMS, often mutant in the RAS pathway. Risk stratifications of RMS patients determine different prognostic groups and related therapeutic treatment. Current multimodal therapeutic strategies involve surgery, chemotherapy (CHT) and radiotherapy (RT), but despite the deeper knowledge of response mechanisms underpinning CHT treatment and the technological improvements that characterize RT, local failures and recurrence frequently occur. This review sums up the RMS classification and the management of RMS patients, with special attention to RT treatment and possible radiosensitizing strategies for RMS tumors. Indeed, RMS radioresistance is a clinical problem and further studies aimed at dissecting radioresistant molecular mechanisms are needed to identify specific targets to hit, thus improving RT-induced cytotoxicity.

Radioresistance in rhabdomyosarcomas: Much more than a question of dose.

Management of rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, frequently accounting the genitourinary tract is complex and requires a multimodal therapy. In particular, as a consequence of the advancement in dose conformity technology, radiation therapy (RT) has now become the standard therapeutic option for patients with RMS. In the clinical practice, dose and timing of RT are adjusted on the basis of patients' risk stratification to reduce late toxicity and side effects on normal tissues. However, despite the substantial improvement in cure rates, local failure and recurrence frequently occur. In this review, we summarize the general principles of the treatment of RMS, focusing on RT, and the main molecular pathways and specific proteins involved into radioresistance in RMS tumors. Specifically, we focused on DNA damage/repair, reactive oxygen species, cancer stem cells, and epigenetic modifications that have been reported in the context of RMS neoplasia in both in vitro and in vivo studies. The precise elucidation of the radioresistance-related molecular mechanisms is of pivotal importance to set up new more effective and tolerable combined therapeutic approaches that can radiosensitize cancer cells to finally ameliorate the overall survival of patients with RMS, especially for the most aggressive subtypes.

Hyperactive Akt1 Signaling Increases Tumor Progression and DNA Repair in Embryonal Rhabdomyosarcoma RD Line and Confers Susceptibility to Glycolysis and Mevalonate Pathway Inhibitors.

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.

Essential Amino Acids-Rich Diet Decreased Adipose Tissue Storage in Adult Mice: A Preliminary Histopathological Study.

BACKGROUND: Excess body adipose tissue accumulation is a common and growing health problem caused by an unbalanced diet and/or junk food. Although the effects of dietary fat and glucose on lipid metabolism regulation are well known, those of essential amino acids (EAAs) have been poorly investigated. Our aim was to study the influence of a special diet containing all EAAs on retroperitoneal white adipose tissue (rpWAT) and interscapular brown adipose tissue (BAT) of mice. METHODS: Two groups of male Balb/C mice were used. The first was fed with a standard diet. The second was fed with an EAAs-rich diet (EAARD). After 3 weeks, rpWAT and BAT were removed and prepared for subsequent immunohistochemical analysis. RESULTS: EAARD, although consumed significantly less, moderately reduced body weight and BAT, but caused a massive reduction in rpWAT. Conversely, the triceps muscle increased in mass. In rpWAT, the size of adipocytes was very small, with increases in leptin, adiponectin and IL-6 immunostaining. In BAT, there was a reduction in lipid droplet size and a simultaneous increase in UCP-1 and SIRT-3. CONCLUSIONS: A diet containing a balanced mixture of free EAA may modulate body adiposity in mice, promoting increased thermogenesis.

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype.

PURPOSE: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). METHODS: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. RESULTS: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. CONCLUSION: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Caveolin-1 promotes radioresistance in rhabdomyosarcoma through increased oxidative stress protection and DNA repair.

The aim of this work was to investigate whether Caveolin-1 (Cav-1), a membrane scaffolding protein widely implicated in cancer, may play a role in radiation response in rhabdomyosarcoma (RMS), a pediatric soft tissue tumor. For this purpose, we employed human RD cells in which Cav-1 expression was stably increased via gene transfection. After radiation treatment, we observed that Cav-1 limited cell cycle arrest in the G2/M phase and enhanced resistance to cell senescence and apoptosis via reduction of p21Cip1/Waf1, p16INK4a and Caspase-3 cleavage. After radiotherapy, Cav-1-mediated cell radioresistance was characterized by low accumulation of H2AX foci, as confirmed by Comet assay, marked neutralization of reactive oxygen species (ROS) and enhanced DNA repair via activation of ATM, Ku70/80 complex and DNA-PK. We found that Cav-1-overexpressing RD cells, already under basal conditions, had higher glutathione (GSH) content and greater catalase expression, which conferred protection against acute treatment with hydrogen peroxide. Furthermore, pre-treatment of Cav-1-overexpressing cells with PP2 or LY294002 compounds restored the sensitivity to radiation treatment, indicating a role for Src-kinases and Akt pathways in Cav-1-mediated radioresistance. These findings were confirmed using radioresistant RD and RH30 lines generated by hypofractionated radiotherapy protocol, which showed marked increase of Cav-1, catalase and Akt, and sensitivity to PP2 and LY294002 treatment. In conclusion, these data suggest that concerted activity of Cav-1 and catalase, in cooperation with activation of Src-kinase and Akt pathways, may represent a network of vital mechanisms that allow irradiated RMS cells to evade cell death induced by oxidative stress and DNA damage.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Modulating the dose-rate differently affects the responsiveness of human epithelial prostate- and mesenchymal rhabdomyosarcoma-cancer cell line to radiation.

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing ß-galactosidase (SA-ß-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.

The Antitumor Didox Acts as an Iron Chelator in Hepatocellular Carcinoma Cells.

Ribonucleotide reductase (RR) is the rate-limiting enzyme that controls the deoxynucleotide triphosphate synthesis and it is an important target of cancer treatment, since it is expressed in tumor cells in proportion to their proliferation rate, their invasiveness and poor prognosis. Didox, a derivative of hydroxyurea (HU), is one of the most potent pharmaceutical inhibitors of this enzyme, with low in vivo side effects. It inhibits the activity of the subunit RRM2 and deoxyribonucleotides (dNTPs) synthesis, and it seems to show iron-chelating activity. In the present work, we mainly investigated the iron-chelating properties of didox using the HA22T/VGH cell line, as a model of hepatocellular carcinoma (HCC). We confirmed that didox induced cell death and that this effect was suppressed by iron supplementation. Interestingly, cell treatments with didox caused changes of cellular iron content, TfR1 and ferritin levels comparable to those caused by the iron chelators, deferoxamine (DFO) and deferiprone (DFP). Chemical studies showed that didox has an affinity binding to Fe3+ comparable to that of DFO and DFP, although with slower kinetic. Structural modeling indicated that didox is a bidentated iron chelator with two theoretical possible positions for the binding and among them that with the two hydroxyls of the catechol group acting as ligands is the more likely one. The iron chelating property of didox may contribute to its antitumor activity not only blocking the formation of the tyrosil radical on Tyr122 (such as HU) on RRM2 (essential for its activity) but also sequestering the iron needed by this enzyme and to the cell proliferation.

Animal models of well-differentiated/dedifferentiated liposarcoma: utility and limitations.

Liposarcoma is a malignant neoplasm of fat tissue. Well-differentiated and dedifferentiated liposarcoma (WDL/DDL) represent the two most clinically observed histotypes occurring in middle-aged to older adults, particularly within the retroperitoneum or extremities. WDL/DDL are thought to represent the broad spectrum of one disease, as they are both associated with the amplification in the chromosomal 12q13-15 region that causes MDM2 and CDK4 overexpression, the most useful predictor for liposarcoma diagnosis. In comparison to WDL, DDL contains additional genetic abnormalities, principally coamplifications of 1p32 and 6q23, that increase recurrence and metastatic rate. In this review, we discuss the xenograft and transgenic animal models generated for studying progression of WDL/DDL, highlighting utilities and pitfalls in such approaches that can facilitate or impede the development of new therapies.

Pro-differentiating and radiosensitizing effects of inhibiting HDACs by PXD-101 (Belinostat) in in vitro and in vivo models of human rhabdomyosarcoma cell lines.

This study describes the in vitro and in vivo activity of PXD-101 (Belinostat), a novel hydroxamic acid-type pan-HDACs inhibitor characterized by a larger safety and efficacy, on myogenic-derived PAX3/FOXO1 fusion protein positive (RH30) or negative (RD) expressing rhabdomyosarcoma (RMS) cell lines. PXD-101 at low doses efficiently inhibited HDACs activity and counteracted the transformed phenotype of RMS by inducing growth arrest and apoptosis, affecting cancer stem cells population and inducing differentiation in RD. Notably, PXD-101 induced oxidative stress promoting DNA damages and affected the ability of RMS to assemble mitotic spindle. PXD-101 radiosensitized by inducing G2 cell cycle growth arrest, enhancing the radiation's ability to induce ROS accumulation and compromising both the ability of RMS to detoxify from ROS and to repair DNA damage. PXD-101 transcriptionally and post-transcriptionally affected c-Myc expression, key master regulator of rhabdomyosarcomagenesis and RMS radioresistance. All in vitro data were corroborated by in vivo experiments showing the cytostatic effects of PXD-101 when used alone and at low dose and its ability to promote the RT-induced killing of RMS. Taken together, our data confirm that altered HDACs activity plays a key role in RMS genesis and suggest PXD-101 as a valid therapeutic strategy particularly in combination with RT.

Correction to: Histone deacetylase inhibitor ITF2357 (givinostat) reverts transformed phenotype and counteracts stemness in in vitro and in vivo models of human glioblastoma.

In the original publication, the 6th author's first name and the last name was inverted and incorrectly published. The correct author name is Letizia Ferella.

Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways.

BACKGROUND Urocortin (Ucn) is a member of the hypothalamic corticotrophin-releasing factor family and has been shown to reduce cell death in the heart caused by ischemia/reperfusion (I/R) injury. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor known to function as a pro-survival and anti-apoptotic factor, whose activation depends on a variety of cytokines, including IL-6. A recent study demonstrated that urocortin induced IL-6 release from cardiomyocytes in a CRF-R2-dependent manner, suggesting a possible link between CRF-R2 stimulation and STAT3 activation. MATERIAL AND METHODS Experimental work was carried out in HL-1 cardiac myocytes exposed to serum starvation for 16-24 h. RESULTS Ucn stimulation led to IL-6 expression and release from mouse atrial HL-1 cardiomyocytes. Ucn treatment led to rapid phosphorylation of JAK2, which was blocked by the protein synthesis inhibitor cycloheximide or the JAK inhibitor AG490. Urocortin treatment induced STAT3 phosphorylation at Y705 and S727 through transactivation of JAK2 in an IL-6-dependent manner, but had no effect on STAT1 activity. Kinase inhibition experiments revealed that urocortin induces STAT3 S727 phosphorylation through ERK1/2 and Y705 phosphorylation through Src tyrosine kinase. In line with this finding, urocortin failed to induce phosphorylation of Y705 residue in SYF cells bearing null mutation of Src, while phosphorylation of S727 residue was unchanged. CONCLUSIONS Here, we have shown that Ucn induces activation of STAT3 through diverging signaling pathways. Full understanding of these signaling pathways will help fully exploit the cardioprotective properties of endogenous and exogenous Ucn.

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.