IDH mutations in G2-3 conventional central bone chondrosarcoma: a mono institutional experience.

BACKGROUND: Heterozygous isocitrate dehydrogenase (IDH) mutations occur in about half of conventional central bone chondrosarcomas (CCBC). Aim of this study was to assess the frequency and prognostic impact of IDH mutations in high grade CCBC patients. METHODS: 64 patients with G2 and G3 CCBC were included. DNA extraction, PCR amplification of IDH1/2 exon 4s, and sequencing analysis with Sanger were performed. RESULTS: IDH mutations were detected in 24/54 patients (44%): IDH1 in 18, IDH2 in 4, and both IDH1/2 in 2 patients. The frequency of mutations was 37% in G2 vs. 69% in G3 (p = 0.039), and 100% in three Ollier disease associated chondrosarcoma. 5-year overall survival (OS) at 124 months (range 1-166) was 51%, with no significant difference based on the IDH mutational status: 61% in IDHmut vs. 44% in IDH wild type (IDHwt). The 5-year relapse free survival (RFS) was 33% (95% CI:10-57) for IDHmut vs. 57% (95%CI: 30-77) for IDHwt. Progression free survival (PFS) was 25% (95%CI:1-65) IDHmut vs. 16% (95%CI: 0.7-52) IDHwt. 55% (5/9) of IDHmut G2 became higher grade at the recurrence, as compared with 25% (3/12) of G2 IDHwt. CONCLUSIONS: This study shows a higher frequency of IDH mutations in G3 CCBC as compared with G2. No significant differences in OS, RFS, and PFS by mutational status were detected. After relapse, a higher rate of G3 for IDH mutated CCBC was observed.

Irinotecan and temozolomide in recurrent Ewing sarcoma: an analysis in 51 adult and pediatric patients.

BACKGROUND: Data on temozolomide (TEM) and irinotecan (IRI) activity in recurrent Ewing sarcoma (EWS), especially in adult patients, are limited. METHODS: Patients receiving TEM 100 mg/m2/day oral, and IRI 40 mg/m2/day intravenous, days 1-5, every 21 days, were included in this multi-institutional retrospective study. Disease control rate (DCR) [overall response rate (ORR) [complete response (CR) + partial response (PR)] + stable disease (SD)], 6-months progression-free survival (6-mos PFS) and 1-year overall survival (OS) were assessed. RESULTS: The median age of the 51 patients was 21 years (range 3-65 years): 34 patients (66%) were adults (≥18 years of age), 24 (48%) had ECOG 1 and 35 (69%) were presented with multiple site recurrence. TEMIRI was used at first relapse/progression in 13 (25%) patients, while the remainder received TEMIRI for second or greater relapse/progression. Fourteen (27%) patients had received prior myeloablative therapy with busulfan and melphalan. We observed five (10%) CR, 12 (24%) PR and 19 (37%) SD, with a DCR of 71%. 6-mos PFS was 49% (95% CI 35-63) and it was significantly influenced by ECOG (6-mos PFS 64% [95% CI 45-83] for ECOG 0, 34% [95% CI 14-54] for ECOG ≥1; p = .006) and LDH (6-mos PFS 62% [95% CI 44-79] for normal LDH, 22% [95% CI 3-42] for high LDH; p = .02), with no difference according to line of treatment, age and metastatic pattern. One-year OS was 55% (95% CI 39-70), with RECIST response (p = .001) and ECOG (p = .0002) independently associated with outcome. Grade 3 and 4 toxicity included neutropenia in 12% of patients, thrombocytopenia in 4%, diarrhea in 4%. CONCLUSIONS: This series confirms the activity of TEMIRI in both adults and pediatric patients. This schedule offers a 71% DCR, independently of the line of chemotherapy. Predictive factors of response are ECOG and LDH.

Identification of genetic variants in pharmacokinetic genes associated with Ewing Sarcoma treatment outcome.

BACKGROUND: Despite the effectiveness of current treatment protocols for Ewing sarcoma (ES), many patients still experience relapse, and survival following recurrence is <15%. We aimed to identify genetic variants that predict treatment outcome in children diagnosed with ES. PATIENTS AND METHODS: We carried out a pharmacogenetic study of 384 single-nucleotide polymorphisms (SNPs) in 24 key transport or metabolism genes relevant to drugs used to treat in pediatric patients (<30 years) with histologically confirmed ES. We studied the association of genotypes with tumor response and overall survival (OS) in a discovery cohort of 106 Spanish children, with replication in a second cohort of 389 pediatric patients from across Europe. RESULTS: We identified associations with OS (P < 0.05) for three SNPs in the Spanish cohort that were replicated in the European cohort. The strongest association observed was with rs7190447, located in the ATP-binding cassette subfamily C member 6 (ABCC6) gene [discovery: hazard ratio (HR) = 14.30, 95% confidence interval (CI) = 1.53-134, P = 0.020; replication: HR = 9.28, 95% CI = 2.20-39.2, P = 0.0024] and its correlated SNP rs7192303, which was predicted to have a plausible regulatory function. We also replicated associations with rs4148737 in the ATP-binding cassette subfamily B member 1 (ABCB1) gene (discovery: HR = 2.96, 95% CI = 1.08-8.10, P = 0.034; replication: HR = 1.60, 95% CI = 1.05-2.44, P = 0.029), which we have previously found to be associated with poorer OS in pediatric osteosarcoma patients, and rs11188147 in cytochrome P450 family 2 subfamily C member 8 gene (CYP2C8) (discovery : HR = 2.49, 95% CI = 1.06-5.87, P = 0.037; replication: HR = 1.77, 95% CI = 1.06-2.96, P = 0.030), an enzyme involved in the oxidative metabolism of the ES chemotherapeutic agents cyclophosphamide and ifosfamide. None of the associations with tumor response were replicated. CONCLUSION: Using an integrated pathway-based approach, we identified polymorphisms in ABCC6, ABCB1 and CYP2C8 associated with OS. These associations were replicated in a large independent cohort, highlighting the importance of pharmacokinetic genes as prognostic markers in ES.

CD99 regulates neural differentiation of Ewing sarcoma cells through miR-34a-Notch-mediated control of NF-κB signaling.

Sarcomas are mesenchymal tumors characterized by blocked differentiation process. In Ewing sarcoma (EWS) both CD99 and EWS-FLI1 concur to oncogenesis and inhibition of differentiation. Here, we demonstrate that uncoupling CD99 from EWS-FLI1 by silencing the former, nuclear factor-κB (NF-κB) signaling is inhibited and the neural differentiation program is re-established. NF-κB inhibition passes through miR-34a-mediated repression of Notch pathway. CD99 counteracts EWS-FLI1 in controlling NF-κB signaling through the miR-34a, which is increased and secreted into exosomes released by CD99-silenced EWS cells. Delivery of exosomes from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells through miR-34a inhibition of Notch-NF-κB signaling. Notably, even the partial delivery of CD99 small interfering RNA may have a broad effect on the entire tumor cell population owing to the spread operated by their miR-34a-enriched exosomes, a feature opening to a new therapeutic option.

CXCL14, CXCR7 expression and CXCR4 splice variant ratio associate with survival and metastases in Ewing sarcoma patients.

PURPOSE: Ewing sarcoma (EWS) is the second most common sarcoma of bone in children and young adults. Patients with disseminated disease at diagnosis or early relapse have a poor prognosis. Our goal was to identify novel predictive biomarkers for these patients, focusing on chemokines, specifically genes involved in the CXCR4-pathway because of their established role in metastasis and tumour growth. METHODS: Total RNA isolated from therapy-naïve tumour samples (n=18; panel I) and cell lines (n=21) was used to study expression of CXCR4-pathway related genes and CXCR4 splice variants (CXCR4-2: Small and CXCR4-1: Large) by RT-Q-PCR. Expression levels were correlated to overall survival (OS) and event free survival (EFS). Study results were validated in an independent series of 26 tumour samples (panel II) from therapy-naïve tumour samples. RESULTS: CXCL12, CXCR4, CXCR7 and CXCL14 were expressed and high CXCR7 and CXCL14 expression showed a positive correlation with EFS and OS and a negative correlation with metastasis development. Both splice variants CXCR4 were expressed in cell lines and tumour samples and CXCR4-1/CXCR4-2 ratio was significantly higher in tumour samples compared to cell lines and correlated with an improved EFS and OS. The results from the test panel were validated in an independent sample panel. CONCLUSIONS: We identified a set of genes involved in CXCR4 signalling that may be used as a marker to predict survival and metastasis development in Ewing sarcoma.

Integrated approaches to miRNAs target definition: time-series analysis in an osteosarcoma differentiative model.

BACKGROUND: microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate. METHODS: Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed. RESULTS: We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs. CONCLUSIONS: These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.

Prognostic significance of miR-34a in Ewing sarcoma is associated with cyclin D1 and ki-67 expression.

BACKGROUND: At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients. PATIENTS AND METHODS: Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients. RESULTS: High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67. CONCLUSIONS: By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity.

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and ß-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and ß-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma.

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

Nucleotide excision repair gene variants and association with survival in osteosarcoma patients treated with neoadjuvant chemotherapy.

The aim of this study was to investigate the role of common polymorphisms in the nucleotide excision repair pathway genes in the tumorigenesis of osteosarcoma and in the response to DNA damaging therapies, such as cisplatin-based neoadjuvant therapy. Excision repair cross-complementing (ERCC) group 2 (XPD; rs13181 and rs1799793), group 5 (XPG; rs17655) and group 1 (XPA; rs3212986 and rs11615) polymorphisms were analyzed in a group of 130 homogenously treated patients with high-grade osteosarcoma, for association with event-free survival (EFS), using the Kaplan-Meier plots and log-rank test. A positive association was observed between both XPD single-nucleotide polymorphisms and an increased EFS (hazards ratio (HR) = 0.34, 95% confidence interval (CI) 0.12-0.98 and HR = 0.19, 95% CI 0.05-0.77, respectively). We had also performed a case-control study for relative risk to develop osteosarcoma. Patients carrying at least one variant allele of XPD rs1799793 had a reduced risk of developing osteosarcoma, compared with wild-type patients (odds ratio = 0.55, 95% CI 0.36-0.84). This study suggests that XPD rs1799793 could be a marker of osteosarcoma associated with features conferring either a better prognosis or a better outcome after platinum therapy, or both.

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling.

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewing's sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle.

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Targeted therapies in bone sarcomas.

The treatment of sarcoma urgently requires new, innovative therapeutic strategies. The most recent improvements in the cure of patients with localized disease have been achieved by dose-intensification, in turn paying the price of acute severe toxicity and secondary malignancies. Keeping side-effects to a minimum is an important goal for pediatric patients and this may be achieved by combining standard cytotoxic chemotherapy with targeted approaches. In addition, after first-line therapy, very limited treatment options remain for patients with disease progression, who, like patients with metastasis at diagnosis, are in urgent need of more effective drugs. The present review highlights key examples of target identification in bone sarcomas, including chimeric oncoproteins, insulin-like growth factor receptor (IGF-IR), and tumor/microenvironment interactions. The review identifies questions and concerns that still need to be addressed before proceeding to safe clinical trials with agents against these promising new targets.

Clinical impact of the methotrexate resistance-associated genes C-MYC and dihydrofolate reductase (DHFR) in high-grade osteosarcoma.

BACKGROUND: Aims of this study were the validation of C-MYC involvement in methotrexate (MTX) resistance and the assessment of clinical impact of C-MYC and dihydrofolate reductase (DHFR) in osteosarcoma (OS). MATERIALS AND METHODS: The involvement of C-MYC in MTX resistance was validated with an antisense approach. C-MYC and DHFR protein levels at diagnosis were assessed by immunohistochemistry on series of patients treated with either a MTX-based protocol (IOR/OS-1; 72 patients) or with a standard four-drug regimen (ISG/SSG 1; 61 patients). RESULTS: Down-regulation of C-MYC significantly decreased the MTX resistance level of OS cells, demonstrating its causal involvement in this phenomenon. In clinical samples, a worse outcome was associated with increased levels of DHFR and C-MYC at diagnosis in the IOR/OS-1 patients and of C-MYC in the ISG/SSG 1 patients. CONCLUSIONS: Meanwhile the adverse clinical impact of DHFR overexpression appeared to be closely related to the relevance of MTX in the chemotherapeutic protocol, that of C-MYC overexpression was more general and not strictly MTX related. The assessment of C-MYC and DHFR at diagnosis, together with that of other known prognostic markers, can be considered for an early identification of subgroups of OS patients with higher risk of adverse outcome.

Microdeletions in 9p21.3 induce false negative results in CDKN2A FISH analysis of Ewing sarcoma.

Deletion of the CDKN2A locus at 9p21.3 has been reported to be a poor prognostic sign in the Ewing sarcoma family of tumours. In clinical applications CDKN2A deletion is primarily detected using fluorescent in situ hybridisation (FISH) with a commercial probe, size approximately 190 kb. Due to limitations in resolution, FISH analysis may fail to detect microdeletions smaller than 190 kb. In the present study, we performed 44K array comparative genomic hybridisation (CGH) on eleven Ewing sarcoma cell lines and 26 tissue samples in order to define the sizes of 9p21.3 deletions. Microarray CGH analysis revealed 9p21.3 deletions encompassing the CDKN2A locus in eight cell lines (73%) and in six tumours (23%). In four cases (two cell lines and two tissue samples) the deletion was less than 190 kb in size. In one cell line sample, we detected a microdeletion of approximately 58 kb in 9p21.3 harbouring the CDKN2A locus. We confirmed this result using 244K microarray CGH and TaqMan quantitative RT-PCR analysis and further performed FISH analysis on this cell line sample. Here, we show that CDKN2A FISH analysis can give false negative results in cases with small microdeletions. Our results suggest that new and more accurate FISH methods should be developed for detection of deletions in the CDKN2A locus.

CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.

CD99 gene encodes two distinct proteins, produced by alternative splicing of CD99 gene transcript. Full-length CD99 isoform (CD99wt) is formed by an extracellular domain, followed by a transmembrane domain and a 36 amino-acid intracytoplasmic domain, which is partially deleted in the truncated, short form (CD99sh). A differential expression of these two CD99 molecules can lead to distinct functional outcomes in lymphocytes. To investigate the functional effects of CD99 molecules on malignancy, forced overexpression of the two CD99 isoforms was induced in osteosarcoma and prostate cancer cells. The two isoforms exhibited opposite functions: the major form dramatically inhibits anchorage-independent growth, anoikis resistance, migration and metastasis, whereas the CD99sh remarkably favours the phenomena. A mechanistic analysis of CD99-transfected osteosarcoma cells points to involvement of c-Src family kinase activity in regulating CD99 functions in malignancy. Ser168 residue of CD99 plays a pivotal role in the reversion of the malignant phenotype. Our findings highlight the involvement of CD99 in crucial processes of cancer malignancy, serving as a curtain raiser for this, so far neglected molecule. In addition, a dualistic role for the two CD99 isoforms was shown in agreement with what was observed for other cell adhesion molecules.

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation.

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.

Prognostic and therapeutic targets in the Ewing's family of tumors (PROTHETS).

ProTheTs is a specific targeted research project (STREP) funded by the European Union in the 6th Framework Program (FP6), area: "LSH-2002-2.2.0-8 Translational research on promising predictive and prognostic markers", contract n. LSHC-CT-2004-503036. The research will concentrate on translating the knowledge being created by genomics and other fields of basic research into applications that improve clinical practice and public health.

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.