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Simple Summary: Synovial sarcoma (SyS) is a rare malignant soft tissue sarcoma bearing the chromosomal translocation t(X;18), which encodes the fusion oncoprotein SS18::SSX. More than 80% of the patients, mainly young in age, are initially diagnosed with localized disease with a 5-year survival rate of 70–80%. Metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease shows a poor prognosis with a 5-year survival rate below 10%, representing an urgent clinical issue. This review will focus on: (i) current front-line therapies; (ii) alternative treatments in second line and beyond settings; and (iii) new epigenetic and immunological strategies. The improved understanding of the SyS molecular biology coupled with the recent development of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells, is offering new promising tools. Clinical trial results underline the need for accurate patient selection based on genetic and tumor immune microenvironment signatures. Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70–80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement. [ABSTRACT FROM AUTHOR]

Background: The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. Methods: The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. Results: The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD–YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. Conclusions: We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination. [ABSTRACT FROM AUTHOR]

The cell surface molecule CD99 has gained interest because of its involvement in regulating cell differentiation and adhesion/migration of immune and tumor cells. However, the molecule plays an intriguing and dual role in different cell types. In particular, it acts as a requirement for cell malignancy or as an oncosuppressor in tumors. In addition, the gene encodes for two different isoforms, which also act in opposition inside the same cell. This review highlights key studies focusing on the dual role of CD99 and its isoforms and discusses major critical issues, challenges, and strategies for overcoming those challenges. The review specifically underscores the properties that make the molecule an attractive therapeutic target and identifies new relationships and areas of study that may be exploited. The elucidation of the spatial and temporal control of the expression of CD99 in normal and tumor cells is required to obtain a full appreciation of this molecule and its signaling. [ABSTRACT FROM AUTHOR]

Background: Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin. Materials and Methods: In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples. Results: We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99. Conclusions: In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that targeting glutathione-S-transferase (GST) isoenzymes may be a promising novel strategy to improve the efficacy of conventional chemotherapy in the three most common musculoskeletal tumours: osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma. By using a panel of 15 drug-sensitive and drug-resistant human osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma cell lines, the efficay of the GST-targeting agent 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has been assessed and related to GST isoenzymes expression (namely GSTP1, GSTA1, GSTM1, and MGST). NBDHEX showed a relevant in vitro activity on all cell lines, including the drug-resistant ones and those with higher GSTs levels. The in vitro activity of NBDHEX was mostly related to cytostatic effects, with a less evident apoptotic induction. NBDHEX positively interacted with doxorubicin, vincristine, cisplatin but showed antagonistic effects with methotrexate. In vivo studies confirmed the cytostatic efficay of NBDHEX and its positive interaction with vincristine in Ewing's sarcoma cells, and also indicated a positive effect against the metastatisation of osteosarcoma cells. The whole body of evidence found in this study indicated that targeting GSTs in osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma may be an interesting new therapeutic option, which can be considered for patients who are scarcely responsive to conventional regimens. [ABSTRACT FROM AUTHOR]

Abstract: Aims: The role of IGF system in the pathogenesis of Ewing’s sarcoma (EWS) is well-documented. However, still little information is available about the value of IGF system components as indicators of prognosis. Understanding the clinical role for IGF system in EWS patients may be important because different subtypes of patients have distinct outcome and may require different treatment protocol. We evaluated the expression of insulin-like growth factor (IGF)-receptor (IGF-IR), insulin receptor (IR), IGF-I and some major intracellular mediators (IRS1, p-ERK) in specimens from EWS patients with primary localised untreated tumours. Patients and methods: 290 samples were used for immunohistochemistry studies; 57 samples for Real-Time PCR studies; and serum of 67 patients for ELISA. Results: IGF-IR and IR are expressed in virtually all EWS tumours. Usually both the receptors are present in the same tumour but when one receptor is lacking the other one is always present. Evaluation of IGF-IR, IR and IGF-I with quantitative methods may discriminate differential levels of expression with influences on the patients’ outcome. High expression of IGF-IR, IR and IGF-I mRNAs is significantly associated with more favourable clinical outcomes. Higher circulating levels of IGF-I also correlated with lower risk to disease progression and death. Conclusions: Overall, our clinical data are in contrast with the assumption that higher amounts of IGF/IGF-IR are a surrogate for higher aggressiveness and indicate that in some cancers the transition to frank malignancy and poor treatment responsiveness seem to be associated with a reduction of IGF system activity. [Copyright &y& Elsevier]

Abstract: Aims: The role of IGF system in the pathogenesis of Ewing’s sarcoma (EWS) is well-documented. However, still little information is available about the value of IGF system components as indicators of prognosis. Understanding the clinical role for IGF system in EWS patients may be important because different subtypes of patients have distinct outcome and may require different treatment protocol. We evaluated the expression of insulin-like growth factor (IGF)-receptor (IGF-IR), insulin receptor (IR), IGF-I and some major intracellular mediators (IRS1, p-ERK) in specimens from EWS patients with primary localised untreated tumours. Patients and methods: 290 samples were used for immunohistochemistry studies; 57 samples for Real-Time PCR studies; and serum of 67 patients for ELISA. Results: IGF-IR and IR are expressed in virtually all EWS tumours. Usually both the receptors are present in the same tumour but when one receptor is lacking the other one is always present. Evaluation of IGF-IR, IR and IGF-I with quantitative methods may discriminate differential levels of expression with influences on the patients’ outcome. High expression of IGF-IR, IR and IGF-I mRNAs is significantly associated with more favourable clinical outcomes. Higher circulating levels of IGF-I also correlated with lower risk to disease progression and death. Conclusions: Overall, our clinical data are in contrast with the assumption that higher amounts of IGF/IGF-IR are a surrogate for higher aggressiveness and indicate that in some cancers the transition to frank malignancy and poor treatment responsiveness seem to be associated with a reduction of IGF system activity. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease. [ABSTRACT FROM AUTHOR]

The relationship between P-glycoprotein expression and malignancy is controversial. We have recently found that, in osteosarcoma, multidrug resistance (MDR) is associated with a less aggressive behavior, both in vitro and in clinical settings. In this study, we evaluated whether P-glycoprotein overexpression has a cause-effect relationship with the reduced metastatic potential of MDR cells, or rather reflects a more complex phenotype. MDR1 gene-transfected osteosarcoma cell clones, showing different levels of P-glycoprotein expression, were analysed for their in vitro characteristics and their tumorigenic and metastatic ability in athymic mice. Apart from the different levels of P-glycoprotein, no significant change in the expression of surface antigens or in the differentiative features were observed in the MDR1 gene transfectants compared to the parental cell lines or control clones, obtained by transfection with neo gene alone. In contrast to controls, however, MDR1 transfectants showed a significantly lower ability to grow in semi-solid medium and were completely unable to grow and give lung metastases in athymic mice. These findings indicate that P-glycoprotein overexpression is causally associated with a low malignant potential of osteosarcoma cells, and open new insights on the role and functions of P-glycoprotein activity. [ABSTRACT FROM AUTHOR]

IL-1 family members have multiple pleiotropic functions affecting various tissues and cells, including the regulation of the immune response, hematopoietic homeostasis, bone remodeling, neuronal physiology, and synaptic plasticity. Many of these activities are involved in various pathological processes and immunological disorders, including tumor initiation and progression. Indeed, IL-1 family members have been described to contribute to shaping the tumor microenvironment (TME), determining immune evasion and drug resistance, and to sustain tumor aggressiveness and metastasis. This review addresses the role of IL-1 family members in bone sarcomas, particularly the highly metastatic osteosarcoma (OS) and Ewing sarcoma (EWS), and discusses the IL-1-family-related mechanisms that play a role in bone metastasis development. We also consider the therapeutic implications of targeting IL-1 family members, which have been proposed as (i) relevant targets for anti-tumor and anti-metastatic drugs; (ii) immune checkpoints for immune suppression; and (iii) potential antigens for immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. Methods: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. Results: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. Conclusions: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs. [ABSTRACT FROM AUTHOR]

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research. [ABSTRACT FROM AUTHOR]

Simple Summary: Giant cell tumors of bone (GCTB) are a histologically benign, yet often aggressive, skeletal tumor. Surgery is the only potentially curative treatment option, but may be associated with severe morbidity and loss of function. Denosumab, a monoclonal antibody directed to RANK ligand, is approved for GCTB treatment. Here we report the results of a phase II correlative study on sBTMs (carboxytermnal-crosslinked-telopeptide of type I collagen (s-CTX), alkaline phosphatase (ALP), bone-alkaline phosphatase (bALP), parathyroid hormone (sPTH), and osteocalcin (OCN) in GCTB patients treated with denosumab. The main finding of the present research is that the use of denosumab in patients with GCTB induces the bALP/ALP/OCN and s-CTX reduction, with a concomitant increase in sPTH. High baseline s-CTX identifies a group of patients at higher risk of progression of the disease. Stratification of the GCTB patients based on s-CTX might guide type of surgery and follow-up. Background: Giant cell tumors of bone (GCTB) are osteolytic tumors. Denosumab, a RANK-L inhibitor, is approved for GCTB. Data on serum bone turnover marker (sBTM) changes are lacking. We present a phase II correlative study on sBTMs in GCTB patients treated with denosumab. Methods: All GCTB patients receiving denosumab within a multicentre, open-label, phase 2 study were enrolled. Serum levels of carboxyterminal-crosslinked-telopeptide of type I collagen (s-CTX), alkaline phosphatase (ALP), bone-alkaline phosphatase (bALP), parathyroid hormone (sPTH), and osteocalcin (OCN) were prospectively assessed (baseline, T0, 3 months, T1, 6 months, T2). The primary endpoint was assessment of sBTM changes after denosumab; the secondary endpoints were disease-free survival (DFS) and sBTM correlation. Results: In 54 cases, sBTMs decreased during denosumab treatment except for sPTH. With a median follow-up of 59 months, 3-year DFS was 65% (%CI 52–79), with a significantly worse outcome for patients with high (≥500 UI/mL) s-CTX at baseline, as compared to low s-CTX (<500 UI/mL) (3-year DFS for high CTX 45% (95%CI 23–67) vs. 75% (95%CI 59–91) for low s-CTX. Higher median ALP and s-CTX were found for patients with tumor size ≥ 5 cm (p = 0.0512; p = 0.0589). Conclusion: Denosumab induces ALP/OCN and s-CTX reduction. High baseline s-CTX identifies a group of patients at higher risk of progression of the disease. [ABSTRACT FROM AUTHOR]

Metformin, a well-known insulin-sensitizer commonly used for type 2 diabetes therapy, has recently emerged as potentially very attractive drug also in oncology. It is cheap, it is relatively safe and many reports have indicated effects in cancer prevention and therapy. These desirable features are particularly interesting for pediatric sarcomas, a group of rare tumors that have been shown to be dependent on IGF and insulin system for pathogenesis and progression. Metformin exerts anti-mitogenic activity in several cancer histotypes through several molecular mechanisms. In this paper, we analyzed its effects against osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, the three most common pediatric sarcomas. Despite in vitro metformin gave remarkable antiproliferative and chemosensitizing effects both in sensitive and chemoresistant cells, its efficacy was not confirmed against Ewing sarcoma xenografts neither as single agent nor in combination with vincristine. This discrepancy between in vitro and in vivo effects may be due to hypoxia, a common feature of solid tumors. We provide evidences that in hypoxia conditions metformin was not able to activate AMPK and inhibit mTOR signaling, which likely prevents the inhibitory effects of metformin on tumor growth. Thus, although metformin may be considered a useful complement of conventional chemotherapy in normoxia, its therapeutic value in highly hypoxic tumors may be more limited. The impact of hypoxia should be considered when novel therapies are planned for pediatric sarcomas. [ABSTRACT FROM AUTHOR]

Correction: Clinical Epigenetics (2019) 11:68 https://doi.org/10.1186/s13148-019-0663-8 Figure 6 of the original publication [[1]] contained a picture duplication in the U-937 panel resulting from an error while copy/pasting the individual pictures used to prepare the figure. Reference 1 Zwergel C, Schnekenburger M, Sarno F, Battistelli C, Manara MC, Stazi G, Mazzone R, Fioravanti R, Gros C, Ausseil F. Identification of a novel quinoline-based DNA demethylating compound highly potent in cancer cells. [Extracted from the article]

The insulin-like growth factor I receptor (IGF-IR) plays an essential role in the establishment and maintenance of transformed phenotype of Ewing's sarcoma (ES) cells, and interference with the IGF-IR pathways by a neutralizing antibody causes reversal of the malignant potential of this neoplasm. In this paper, we stably transfected an IGF-IR antisense mRNA expression plasmid in an ES cell line to determine the effectiveness of antisense strategies against the in vitro and in viva growth of ES cells. Doxorubicin sensitivity of TC-71 cells expressing antisense targeted to IGF-IR mRNA was also examined. Cells carrying antisense IGF-IR had a reduced expression of the receptor, a modest decrease in cell proliferation, a significant increase in anoikis-induced apoptosis, and a severely reduced ability to form colonies in soft agar. Moreover, TC/AS cells showed a marked reduction in their motility. In vivo, when cells carrying antisense IGF-IR were injected subcutaneously in nude mice, tumor formation was delayed and survival increased. Metastatic ability of ES cells was also significantly reduced. Furthermore, TC/AS clones showed a significantly higher sensitivity to doxorubicin — a major drug in the treatment of ES. These results indicate that inhibiting IGF-IR by antisense strategies may be relevant to the clinical treatment of ES patients by reducing the malignant potential of these cells and enhancing the effectiveness of chemotherapy. [ABSTRACT FROM AUTHOR]

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches. [ABSTRACT FROM AUTHOR]

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. B Correction to: Cellular Oncology b https://doi.org/10.1007/s13402-022-00713-5 In this article an incorrect affiliation had inadvertently been added. [Extracted from the article]

The article presents a study on nuclear shape instability evoked by lamin A deregulation that promotes metastases in ewing sarcoma. Topics include information on Ewing sarcoma (EWS) which is type of bone cancer; presence of metastases at the onset remains the most clinically relevant adverse prognostic factor for EWS; and features of cancer.

Background Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. Methods Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumor efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. Results Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. Conclusions Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation. [ABSTRACT FROM AUTHOR]

Nuclear morphology abnormalities, loss of nuclear lamina integrity and changes in its constitution are known cancer hallmarks [1]. Alterations in nuclear architecture are mediated by nuclear proteins, such as lamin A/C, the main constituent of nuclear lamina, or LINC complex proteins SUN1, SUN2 and nesprins, which mediate a mechanical link between nucleus and cytoskeleton, determining the overall nuclear structural properties [1]. Lamin alterations may decrease nuclear rigidity, promoting invasiveness or increase protection against mechanical forces, such as interstitial pressure within the tumor [2, 3]. It has been observed that the expression of lamin A can be increased, reduced or absent, depending on the type of cancer. However, changes in lamin A levels are often associated with poor prognosis in multiple human cancers [4]. Here, we reported that low LMNA levels are associated with a worse 5-year OS in Ewing Sarcoma (EWS) patients and correlated with a more metastatic phenotype. To gain mechanistic insights into lamin A-dependent pathways in EWS, a pediatric tumor characterized by a high degree of aggressiveness, we focused on the LINC complex proteins, the major lamin A partners in mechanosignaling transduction [5]. We found that SUN1, SUN2, and nesprin 2 were severely mislocalized, due to a lamin A-dependent mechanism, as forced lamin A expression rescued their nuclear localization and SUN1 expression involved in cytoskeleton remodelling, and directly correlated to EWS invasiveness [6]. While in naïve EWS cells, YAP was recruited to the nucleus, lamin A overexpression reduced its nuclear retention, inhibiting its activity. Lamin A rescue of SUN1 levels/localization was associated with significantly lower ROCK2 levels and reduced motility, in agreement with the critical role played by Rho/ROCK2 pathway in EWS migration [7]. Interestingly, we obtained the same effects treating EWS cells with mevinolin, a statin already employed in clinical practice. Mevinolin treatment caused prelamin A accumulation as expected, but also significantly increased LMNA gene and protein expression, rescuing LINC complex localization and pushing the cells in a more differentiated status. Thus, our findings recognize mevinolin, as a tool capable of reducing invasiveness, while triggering neural differentiation, paving the way to new therapeutic options for this very aggressive cancer. [ABSTRACT FROM AUTHOR]

Ewing sarcoma (EWS) is an aggressive bone tumor of uncertain cellular origin. CD99 is a membrane protein that is expressed in most cases of EWS, although its function in the disease is unknown. Here we have shown that endogenous CD99 expression modulates EWS tumor differentiation and malignancy. We determined that knocking down CD99 expression in human EWS cell lines reduced their ability to form tumors and bone metastases when xenografted into immunodeficient mice and diminished their tumorigenic characteristics in vitro. Further, reduction of CD99 expression resulted in neurite outgrowth and increased expression of beta-III tubulin and markers of neural differentiation. Analysis of a panel of human EWS cells revealed an inverse correlation between CD99 and H-neurofilament expression, as well as an inverse correlation between neural differentiation and oncogenic transformation. As knockdown of CD99 also led to an increase in phosphorylation of ERK1/2, we suggest that the CD99-mediated prevention of neural differentiation of EWS occurs through MAPK pathway modulation. Together, these data indicate a new role for CD99 in preventing neural differentiation of EWS cells and suggest that blockade of CD99 or its downstream molecular pathway may be a new therapeutic approach for EWS. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing's sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing's sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms. [ABSTRACT FROM AUTHOR]

Abstract: Since osteosarcoma is a drug-resistant disease, the aim of the present study was to explore the possible interest of therapeutic approaches including nitrogen-containing biphosphonate zoledronic acid using osteosarcoma cell lines with different genetic backgrounds. Parental p53+/pRb+ U2-OS, p53-mutant U2-OS (U2-OS/175) and p53−/pRb− SAOS were sensitive to zoledronic acid with no significant differences in IC50 values. Analysis of cell cycle distribution revealed a time-dependent shifting of U2-OS cells towards G2 phase with cell cycle arrest in G2 phase at 96h of exposure to the compound. Conversely, U2-OS/175 and SAOS cells responded to treatment with transient cell accumulation in S phase up to 48–72h, respectively. Cell lines were exposed to increasing concentrations of cisplatin alone or combined with sub-toxic doses of zoledronic acid. A growth inhibitory effect was seen after combined treatment in U2-OS, otherwise resistant to cisplatin up to 100ng/ml. Zoledronic acid did not efficiently sensitized U2-OS/175 and SAOS to cisplatin, thereby suggesting that different behavior may depend on p53 mutation. This data was confirmed in U2-OS cells where p53 expression was downregulated by RNA interference. Present findings indicate occurrence of sensitization to cisplatin by zoledronic acid in wild-type p53 osteosarcoma cells but not in p53-null cells nor in cells expressing a dominant-negative form of p53, supporting that wild-type p53 is required for synergistic interaction of cisplatin and zoledronic acid. [Copyright &y& Elsevier]

Abstract: CD99 is a 32kDa surface glycoprotein that is involved in the migration of leukocytes, cell–cell adhesion and apoptosis of T cells and Ewing’s sarcoma (ES) cells, two cell types with a high level of CD99 expression. Engagement of the molecule induces a rapid death signal that appears to be related to the level of expression of this antigen. The rapid apoptosis induced by agonistic anti-CD99 monoclonal antibodies is of clinical interest in ES, a tumour for which no new drugs have been described as clearly effective in the last 10 years. In this study, we show that an anti-CD99 monoclonal antibody can be used to advantage in association with doxorubicin. Striking effectiveness was observed against local tumours and metastases. No remarkably toxic effects of anti-CD99 monoclonal antibody were found in bone marrow against blood precursors. These results provide the necessary rationale and support for a novel modality of therapeutic intervention, which may have application in the care of patients with ES. [Copyright &y& Elsevier]

Abstract: The effectiveness of the alkycycline 4-demethoxy-3′-deamino-3′-aziridinyl-4′-methylsulphonyl-daunorubicin (PNU-159548, ladirubicin), a new drug with high antitumour activity against a broad range of neoplasms, was evaluated by using a panel of 32 human osteosarcoma cell lines, including cell lines resistant to doxorubicin, methotrexate, or cisplatin. PNU-159548 resulted to be highly active in all cell lines. No cross-resistance was found with conventional drugs, being PNU-159548 active also in cells resistant to doxorubicin and with a multidrug resistance phenotype (associated with MDR1 gene/P-glycoprotein overexpression), as well as in cells resistant to methotrexate or to cisplatin. Analysis of drug–drug interactions showed that PNU-159548 could be successfully used in combination with all the most important drugs currently used in OS chemotherapy. In fact, the simultaneous administration of PNU-159548 and doxorubicin, methotrexate, or cisplatin produced mostly additive or synergistic effects. Sequential exposure to PNU-159548 followed immediately by doxorubicin, methotrexate, or cisplatin was the most effective sequence of administration, invariably resulting in additive or synergistic effects in both drug-sensitive and drug-resistant osteosarcoma cell lines. In conclusion, the high in vitro effectiveness, the absence of cross-resistance with doxorubicin, methotrexate, or cisplatin and the possibility to be successfully used in combination with these drugs indicate PNU-159548 as a promising candidate to be considered for planning new therapeutic regimens for osteosarcoma patients, who show a decreased response to conventional chemotherapy. [Copyright &y& Elsevier]

Altered expression of CCN3 has been observed in a variety of musculoskeletal tumours, including Ewing's sarcoma (ES). Despite its widespread distribution, very little is known about its biological functions and molecular mechanisms of action. We transfected CCN3 gene into a CCN3-negative ES cell line and analysed the in vitro and in vivo behaviours of stably transfected clones. Forced expression of CCN3 significantly reduced cell proliferation in vitro, growth in anchorage-independent conditions, and tumorigenicity in nude mice. Despite the antiproliferative effect, CCN3-transfected ES cells displayed increased migration and invasion of Matrigel. The decreased expression of α2β1 integrin receptor and the increased amount of cell surface-associated matrix metalloproteinase (MMP)-9 following the expression of CCN3 may be the basis for the increased migratory abilities of transfected cells. Cells lacking α2β1 are less facilitated to have stable anchorage since the predominant collagen extracted from ES tissue is indeed type I collagen, and proMMP-9 was recently found to provide a cellular switch between stationary and migratory ES cell phase. Our findings are in line with those recently obtained in glioblastoma. However, the underlying molecular mechanisms appear to be different, further highlighting the importance of the cellular context in the regulation of function of CCN proteins.Oncogene (2005) 24, 4349–4361. doi:10.1038/sj.onc.1208620 Published online 11 April 2005 [ABSTRACT FROM AUTHOR]

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.Oncogene (2004) 23, 5664-5674. doi:10.1038/sj.onc.1207741 Published online 7 June 2004 [ABSTRACT FROM AUTHOR]

Alkaline phosphatases (ALPs) are a family of cell surface glycoproteins that catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate. Liver/bone/kidney (L/B/K) ALP participates in bone mineralization, but its other physiological and pathological functions remain obscure. In human osteosarcoma, an inverse relationship has been found between cellular L/B/K ALP expression and aggressiveness. To explore this relationship, we employed cDNA microarray technology to characterize and compare the gene expression profile of two U-2 OS osteosarcoma clones with high L/B/K ALP activity (U-2/ALP28 and U-2/ALP40) and one with contrasting characteristics (U-2/ALP23). We identified 79 differentially expressed genes (58 upregulated in U-2/ALP28 and U-2/ALP40 compared to U-2/ALP23). Using GenMAPP/MAPPFinder, we highlighted nine functional groups strictly related to high L/B/K ALP activity, including microtubule-based movement and cell adhesion groups, two functions well related to tumor invasiveness. Notably, cadherin 13 (CDH13) and caveolin 1 (CAV1) genes were upregulated in our cells. Since these two genes are involved in cell–cell adhesion and cell growth, their co-expression with L/B/K ALP could help explain the lower levels of malignancy found in osteosarcoma cells with high L/B/K ALP activity. Although functional studies are needed to better define the role of CDH13 and CAV1 in the malignant behavior of osteosarcoma cells, the data presented here provide an aid to understanding the biological functions of L/B/K ALP in bone tumors. [Copyright &y& Elsevier]

We retrospectively analysed a group of primary tumours from 32 patients with synovial sarcomas of the extremities by using immunohistochemistry on paraffin-embedded tissue samples in order to investigate the prognostic significance of the nuclear accumulation of c-myc and mdm2 proteins. Nuclear positive immunostaining for c-myc or mdm2 proteins were revealed in 9/32 cases (28%) and in 6/30 cases (20%), respectively. Since c-myc protein appears to be a key factor for keeping cells in an active proliferative stage, we also analysed the growth compartment of each tumour by using the MIB1 monoclonal antibody, specific for Ki-67 antigen. A high MIB1 index was found in 8/31 cases (26%) but was not associated either with c-myc or mdm2 nuclear positivity. Analysis of clinical outcome was performed in a subgroup of 27 patients with a minimum follow-up of 24 months. Among the clinicopathologic parameters and the biological markers, only the nuclear accumulation of c-myc was significantly associated with a higher relapse rate (p = 0.03). Accordingly, survival analysis confirmed a trend toward a poor event-free survival rate and a worse outcome in c-myc-positive cases (p = 0.12). These data demonstrate that assessment of c-myc nuclear accumulation can be useful to identify high-risk subsets of patients with synovial sarcoma of the extremities.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: DNA methyltransferases (DNMTs) are epigenetic enzymes involved in embryonic development, cell differentiation, epithelial to mesenchymal transition, and control of gene expression, whose overexpression or enhanced catalytic activity has been widely reported in cancer initiation and progression. To date, two DNMT inhibitors (DNMTi), 5-azacytidine (5-AZA) and 5-aza-2′-deoxycytidine (DAC), are approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia. Nevertheless, they are chemically instable and quite toxic for healthy cells; thus, the discovery of novel DNMTi is urgent. Results: Here, we report the identification of a new quinoline-based molecule, MC3353, as a non-nucleoside inhibitor and downregulator of DNMT. This compound was able, in promoter demethylating assays, to induce enhanced green fluorescence protein (EGFP) gene expression in HCT116 cells and transcription in a cytomegalovirus (CMV) promoter-driven luciferase reporter system in KG-1 cells. Moreover, MC3353 displayed a strong antiproliferative activity when tested on HCT116 colon cancer cells after 48 h of treatment at 0.5 μM. At higher doses, this compound provided a cytotoxic effect in double DNMT knockout HCT116 cells. MC3353 was also screened on a different panel of cancer cells (KG-1 and U-937 acute myeloid leukemia, RAJI Burkitt's lymphoma, PC-3 prostate cancer, and MDA-MB-231 breast cancer), where it arrested cell proliferation and reduced viability after 48 h of treatment with IC50 values ranging from 0.3 to 0.9 μM. Compared to healthy cell models, MC3353 induced apoptosis (e.g., U-937 and KG-1 cells) or necrosis (e.g., RAJI cells) at lower concentrations. Importantly, together with the main DNMT3A enzyme inhibition, MC3353 was also able to downregulate the DNMT3A protein level in selected HCT116 and PC-3 cell lines. Additionally, this compound provided impairment of the epithelial-to-mesenchymal transition (EMT) by inducing E-cadherin while reducing matrix metalloproteinase (MMP2) mRNA and protein levels in PC-3 and HCT116 cells. Last, tested on a panel of primary osteosarcoma cell lines, MC3353 markedly inhibited cell growth with low single-digit micromolar IC50 ranging from 1.1 to 2.4 μM. Interestingly, in Saos-2 osteosarcoma cells, MC3353 induced both expression of genes and mineralized the matrix as evidence of osteosarcoma to osteoblast differentiation. Conclusions: The present work describes MC3353 as a novel DNMTi displaying a stronger in cell demethylating ability than both 5-AZA and DAC, providing re-activation of the silenced ubiquitin C-terminal hydrolase L1 (UCHL1) gene. MC3353 displayed dose- and time-dependent antiproliferative activity in several cancer cell types, inducing cell death and affecting EMT through E-cadherin and MMP2 modulation. In addition, this compound proved efficacy even in primary osteosarcoma cell models, through the modulation of genes involved in osteoblast differentiation. [ABSTRACT FROM AUTHOR]

Background: Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones.Methods: Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts.Results: Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system.Conclusions: Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients. [ABSTRACT FROM AUTHOR]

Background: Increased levels of P-glycoprotein occur in some osteosarcomas. In this study we determined the relation between P-glycoprotein status and outcome in patients with high-grade osteosarcoma. Methods: P-glycoprotein status was determined immunohistochemically in specimens of osteosarcoma of the extremities (stage II) from 92 patients who were treated with surgery and chemotherapy. The P-glycoprotein status was analyzed in relation to the length of event-free survival. Results: The presence of increased levels of P-glycoprotein in the osteosarcoma was significantly associated with a decreased probability of remaining event-free after diagnosis (P = 0.002). In a multivariate analysis, P-glycoprotein status (P = 0.001) and the extent of tumor necrosis after preoperative chemotherapy (P = 0.04) were independent predictors of clinical outcome. The risk of adverse events was increased substantially (rate ratio, 3.37; 95 percent confidence interval, 1.60 to 7.10) among patients with increased levels of P-glycoprotein in tumor cells, as compared with patients who did not have increased levels of P-glycoprotein in tumor cells. Conclusions: In patients with high-grade osteosarcoma treated with surgery and chemotherapy, the presence of increased levels of P-glycoprotein in tumor cells is associated with a significantly increased risk of adverse events and is independent of the extent of necrosis after preoperative chemotherapy. (N Engl J Med 1995;333:1380-5.) [ABSTRACT FROM AUTHOR]