CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis.

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.

NVP-BEZ235 as a new therapeutic option for sarcomas.

PURPOSE: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma). EXPERIMENTAL DESIGN: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewing's sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine. RESULTS: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G(1) phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti-insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects. CONCLUSIONS: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients.

Biological indicators of prognosis in Ewing's sarcoma: an emerging role for lectin galactoside-binding soluble 3 binding protein (LGALS3BP).

Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell-to-cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c-MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event-free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross-validation and meta-analysis, which are indispensable tools for a rare tumor such as EWS.

Prognostic relevance of CCN3 in Ewing sarcoma.

Ewing sarcoma is a highly aggressive malignant bone tumor occurring preferentially in children and young adults. At present, only clinical features, such as patient age, presence of clinically evident metastases at diagnosis, and poor response to neoadjuvant chemotherapy, are widely accepted as prognostic indicators in Ewing sarcoma. In this study, we assessed the prognostic value of CCN3 (Nov), a matricellular protein that play crucial roles in bone formation. Polyclonal antibodies directed against each of the different CCN3 modules were used to identify variant CCN3 proteins in tumors and to draw potential relationships between the expression of these variants and the outcome of patients with Ewing sarcoma. Our results confirmed that expression of the full-length CCN3 in Ewing sarcoma is associated to a worse prognostic. Furthermore, we report a possible relationship between the expression of a CCN3 protein lacking an internal module (von Willebrand factor type C) and sensitivity to radiotherapy. We hypothesize that the increased level of variant CCN3 in the tumor cells reduces their tumorigenic potential and results in better outcome.

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors.

BACKGROUND: Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas. METHODS: Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5-192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest. RESULTS: Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed. CONCLUSION: We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.

Prognostic value of CCN3 in osteosarcoma.

PURPOSE: Osteosarcoma, the most common bone tumor, lacks prognostic markers that could distinguish patients before therapy and drive treatment choices. We assessed the prognostic value of CCN1, CCN2, and CCN3 genes, involved in fundamental biological processes. EXPERIMENTAL DESIGN: Expression of CCN1, CCN2, and CCN3 was measured by quantitative PCR in 45 newly diagnosed osteosarcomas. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations with osteoblastic differentiation and/or drug response genes were assessed in tumor cells using Spearman correlation and Fisher's exact tests. RESULTS: CCN1 and CCN2 expression was associated with genes involved in commitment of mesenchymal stem cells toward osteoblasts and in early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC). Although CCN3 is barely expressed in normal proliferating osteoblasts and mesenchymal stem cells, its expression was generally high in osteosarcoma and its level of expression did not correlate with any specific osteoblastic differentiation genes. High expression of CCN3 significantly correlated with worse prognosis in osteosarcoma. This may be only partly explained by the association with the expression of multidrug resistance-related protein 1 and 4, two ATP-binding cassette transporters that also acted as predictors of worse outcome in our study. CONCLUSIONS: Our study showed temporal and coordinated expression of CCN1, CCN2, and CCN3 genes during osteoblastic differentiation and highlighted significant differences between human normal and osteosarcoma cell differentiation in vitro. CCN1 and CCN2 expression shows no prognostic relevance in osteosarcoma. In contrast, assessment for CCN3 expression levels at diagnosis may represent a useful molecular tool to early identification of patients with different prognosis.

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells.

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells.

Preclinical in vivo study of new insulin-like growth factor-I receptor--specific inhibitor in Ewing's sarcoma.

PURPOSE: Small-molecule insulin-like growth factor-I receptor (IGF-IR)-specific tyrosine kinase inhibitors have been recently proposed as clinically viable approaches to impair IGF-IR functions. NVP-AEW541 seems one of the most promising agents. In this article, we point out its effects against migration, metastasis, vasculogenicity, and angiogenesis of Ewing's sarcoma cells. EXPERIMENTAL DESIGN: In vivo NVP-AEW541 effectiveness was analyzed against TC-71 Ewing's sarcoma growth and bone metastasis after cell inoculation in athymic mice. Activity of the compound against angiogenesis as well as vasculogenesis properties was also considered both in vitro and in xenografts. Serum glucose, urea, transaminase levels, as well as other signs of distress were checked in mice treated with the IGF-IR inhibitor. RESULTS: Significant inhibition of migration, metastasis, vasculogenicity, and angiogenesis was recorded after treatment of Ewing's sarcoma cells with NVP-AEW541. In view of its application and the similarity of insulin receptor and IGF-IR, diabetogenic side effects were considered. We observed a significant decrease of glucose blood serum due to increased glucose uptake at cellular level and an increase in urea concentration. Moreover, an initial weight loss was observed in mice bearing tumors. All these side effects were similarly detected in mice treated with vincristine. After the first days of treatment, all the animals started to grow again. CONCLUSIONS: Our results globally reinforce the idea that IGF-IR inhibitor NVP-AEW541 could have a role in future combined therapies and suggest to pursue a thorough molecular analysis of the metabolic activity of IGF-IR to avoid possible side effects of these inhibitors.

Antitumor activity of the insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors.

Identification of new drugs is strongly needed for sarcomas. Insulin-like growth factor-I receptor (IGF-IR) was found to provide a major contribution to the malignant behavior of these tumors, therefore representing a very promising therapeutic target. In this study, we analyzed the therapeutic potential of a novel kinase inhibitor of IGF-IR, NVP-AEW541, in Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, the three most frequent solid tumors in children and adolescents. NVP-AEW541 inhibits IGF-I-mediated receptor activation and downstream signaling. Ewing's sarcoma cells were generally found to be more sensitive to the effects of this drug compared with rhabdomyosarcoma and osteosarcoma, in agreement with the high dependency of this neoplasm to IGF-IR signaling. NVP-AEW541 induced a G1 cell cycle block in all cells tested, whereas apoptosis was observed only in those cells that show a high level of sensitivity. Concurrent exposure of cells to NVP-AEW541 and other chemotherapeutic agents resulted in positive interactions with vincristine, actinomycin D, and ifosfamide and subadditive effects with doxorubicin and cisplatin. Accordingly, combined treatment with NVP-AEW541 and vincristine significantly inhibited tumor growth of Ewing's sarcoma xenografts in nude mice. Therefore, results encourage inclusion of this drug especially in the treatment of patients with Ewing's sarcoma. For the broadest applicability and best efficacy in sarcomas, NVP-AEW541 may be combined with vincristine, actinomycin D, and ifosfamide, three major drugs in the treatment of sarcomas.

In Ewing's sarcoma CCN3(NOV) inhibits proliferation while promoting migration and invasion of the same cell type.

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 alpha2beta1 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 alpha2beta1 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.