OS Progression Research Articles

RNA-binding protein PUM2 suppresses osteosarcoma progression via partly and competitively binding to STARD13 3'UTR with miRNAs.

This work aims to reveal the roles and related mechanisms of RNA binding protein PUM2 in osteosarcoma progression. Transcriptome analysis based on RNA sequencing data, real-time quantitative PCR (RT-qPCR), and western blot analysis were used to detect the expression of RBPs and miRNAs in OS and normal adjacent tissues, and the correlation between them in OS tissues. RT-qPCR, western blot, cell viability, transwell migration, tumour spheres formation and in vivo tumour formation assays were used to examine the effects of RBP PUM2 on OS progression. Additionally, RNA immunoprecipitation (RIP) assay combined with RNA sequencing was performed to determine the binding site of RBP PUM2 on STARD13 3'UTR. Luciferase reporter and RIP assays were used to confirm the binding of miRNAs or PUM2 on STARD13 3'UTR. PUM2 and STARD13 expression was significantly decreased in OS tissues, and positively correlated. Overexpression of PUM2 or STARD13 3'UTR inhibited OS cells proliferation, migration, and stemness. Mechanistically, PUM2 competitively bound to STARD13 3'UTR with miR-590-3p and miR-9. The inhibition of PUM2 on OS cells progression was attenuated by STARD13 knockdown or related miRNAs overexpression. PUM2 suppresses OS progression via partly and competitively binding to STARD13 3'UTR with miRNAs.

Genetic and clonal dissection of osteosarcoma progression and lung metastasis

Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.

Ovol2 induces mesenchymal-epithelial transition via targeting ZEB1 in osteosarcoma.

PurposeOsteosarcoma (OS) is the most common type of primary solid bone tumor. Ovo-like zinc finger 2 (Ovol2), a zinc finger transcription factor, is a mesenchymal–epithelial transition (MET) driver that induces miR-200 expression in prostate cancer, breast cancer, and hepatocellular carcinoma. However, little is known about the expression and function of MET in sarcomas, including OS. This study investigated the expression and clinicopathological significance of Ovol2 and its effect on MET in OS.Patients and methodsThe Ovol2 expression in the tumor samples from patients with OS was examined using immunohistochemistry (IHC). We then upregulated the Ovol2 expression in MG-63 and SW1353 cells, detected the expression of MET-associated proteins, and observed the effects of Ovol2 on OS cell proliferation, migration, and cytoskeleton reorganization using Cell Counting Kit-8, transwell invasion, and phalloidin dyeing assays, respectively. The correlation between zinc finger E-box-binding homeobox 1 (ZEB1) and Ovol2 was assessed using the luciferase gene reporter assay in the MG-63 and SW1353 cells and IHC in the human OS tissue samples.ResultsThe Ovol2 protein overexpression was related to the clinical grade (P=0.02) and the recurrence and metastasis (P=0.02) of OS. Results of the in vitro experiments showed that Ovol2 overexpression can suppress cell migration and invasion and can regulate the expression levels of MET-associated proteins. Ovol2 suppresses ZEB1 expression by binding to the ZEB1 promoter. Ovol2 is concomitant with a reduced IHC expression of ZEB1 in human OS tissues.ConclusionOvol2 expression is associated with MET in OS cells and suppresses ZEB1 expression and OS progression.

The Value of lncRNA NEAT1 as a Prognostic Factor for Survival of Cancer Outcome: A Meta-Analysis

The present meta-analysis aimed to analyze available data to identify the prognostic role of NEAT1 in multiple carcinomas. A systematic search was performed by using several computerized databases from inception to June 7, 2017. The quantity of the publications was assessed according to MOOSE checklist. Pooled HRs with 95% CI was calculated to summarize the effect. A total of 12 studies with 3,262 cancer patients were pooled in the analysis to evaluate the prognostic value of NEAT1 in multiple tumors. High expression levels of NEAT1 were demonstrated to be associated with poor OS (HR = 1.71, 95%CI: 1.37–2.14, P < 0.001) and tumor progression (III/IV vs. I/II: HR 1.76, 95%CI: 1.40–2.21, P < 0.00001). Subgroup analysis showed that NEAT1 detection method (qRT-PCR) and sample size (more or less than 100) did not alter the predictive value of NEAT1 on OS in various cancers. According to the meta-regression results, the large heterogeneity of meta-analysis may be attributed to the differences of NEAT1 detection method. Furthermore, elevated NEAT1 expression significantly predicted lymph node metastasis (HR: 2.10, 95%CI: 1.32–3.33, P = 0.002) and distant metastasis (HR: 2.80, 95%CI: 1.60–4.91, P = 0.0003) respectively. The results indicate that NEAT1 expression level is a prognostic biomarker for OS and metastasis in general tumors.

Dose-Dense Chemoimmunotherapy Including Early CNS Prophylaxis for High-Risk DLBCL. -Final Analysis from a Nordic Phase II Study (the CHIC trial)

Introduction: Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of early central nervous system (CNS) progression is high. Here we present the final results from a Nordic phase II study, where dose-dense chemoimmunotherapy including early systemic CNS prophylaxis with high dose methotrexate (HD-Mtx), further intensified by intrathecally (IT) administered liposomal cytosine arabinoside (AraC), was given.Patients and methods: Inclusion criteria were age 18-65 years, de novo DLBCL or grade 3B follicular lymphoma without clinical, radiological or cytological signs of CNS involvement, age adjusted IPI 2-3, WHO performance score 0-3, and/or anatomical sites related to increased risk for CNS recurrence (e.g. testis, facial sinuses, orbita). Treatment consisted of two courses of HD-Mtx in combination with R-CHOP14, followed by four courses of R-CHOEP14 and one course of R-HD-AraC. Liposomal AraC was administered IT at courses 1, 3 and 5 (omitted during a period of production halt). Primary end points were failure free survival (FFS; disease progression, discontinuation of protocolled therapy due to toxicity, death from any cause) and CNS progression rate at 18 months. Among the secondary endpoints were the identification of biological risk factors for high risk disease and prognostic role of cerebrospinal fluid (CSF) cytology-/flow cytometry (FC)+for CNS recurrence.Results: Of the accrued 143 patients, 140 met the inclusion criteria and were evaluable for baseline characteristics and primary endpoints. Of these, 132 had a complete set of treatment data. The male/female ratio was 1.7 and the median age 56 years (range 20-64). The majority of the patients had DLBCL (96%), advanced clinical stage (93%), elevated LDH (91%), more than one extranodal site (73%), and B-symptoms (64%). A bulky lesion (>10 cm) was present in 37% of the patients and 11 CSF samples (8%) were FC+. Most patients (n=127, 96%) received a full treatment schedule. Liposomal AraC was given to 81 (61%) and radiotherapy to 39 (30%) patients. Grade 4 infections were observed in 12% of the patients. The frequency of grade 3-4 mucositis as well as gastrointestinal toxicity was 20%, and of grade 3 arachnoiditis 2,5%. Three toxic deaths were observed. In addition, three patients developed AML/MDS and one PML. At the end of treatment, CR/CRu, PR and PD rates were 79%, 17% and 3%, respectively. Of the 120 patients who underwent PET-CT, 92 (77%) achieved a metabolic CR (Deauville score (DS) 1-3). Three patients had primary refractory disease. At a median follow-up of 30 months, additional 14 patients had relapsed, three of them in the CNS (only one had a pre-therapeutic FC+ CSF), and 15 had died. FFS, PFS, OS and CNS progression rates at 30 months were 80%, 83%, 90%, and 2.4%, respectively. PET positivity (DS 4-5) at the end of treatment (p=0.019) and BCL2 expression (p=0.049) were associated with increased risk of progression, whereas other factors, such as molecular subtype (GC versus non-GC), Ki-67 score (≥70%), aaIPI group (2 versus 3), number of extranodal sites, FC-based CSF positivity, and treatment with liposomal AraC did not seem to have significant impact on outcome.Conclusions: Safety profile and final outcome results of the Nordic CHIC trial indicate high response rates, favorable survival, low number of CNS recurrences and manageable toxicity as a result of this CNS targeted intensive therapy schedule. PET response at the end of therapy and selected biological factors identify patients at high risk of progression. DisclosuresLeppa:Roche: Honoraria, Other: Travel expenses, Research Funding; Janssen: Research Funding; Bayer: Research Funding; Mundipharma: Research Funding; Amgen: Research Funding; Takeda: Honoraria, Other: Travel expenses; CTI Life Sciences: Honoraria; Merck: Other: Travel expenses. Joergensen:Amgen: Research Funding; Mundipharma: Research Funding. Mannisto:SOBI: Honoraria; Pfizer: Honoraria; Gilead: Other: Travel expenses; Celgene: Other: Travel expenses; Novartis: Other: Travel expenses; Amgen: Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Roche: Honoraria, Other: Travel expenses. Jerkeman:Gilead: Research Funding; Celgene: Research Funding; Mundipharma: Research Funding; Amgen: Research Funding; Janssen: Research Funding. Holte:Amgen: Research Funding; Mundipharma: Research Funding.

Abstract 589: Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model

Abstract Osteosarcoma (OS) is a malignancy of bone and five-year survival rates are still at a low 20% if metastatic disease is present. Metastatic OSs most frequently metastasize to the lung. TNF-α is a powerful cytokine already shown to be active against many cancers. Nevertheless, TNF-α is highly toxic at clinically relevant concentrations. To overcome this limitation, targeted forms of TNF-α can be used. In this study, targeting is achieved through linking TNF-α to the antibody fragment F8, targeting the EDA-domain of fibronectin. To test the efficacy of F8-TNF-α against OS, we sought to investigate the effects of F8-TNF-α in an orthotopic syngeneic OS model. In addition to evaluating compound efficacy, we also tested whether the route of administration (systemic versus local application) can even further increase treatment efficacy. Ultimately, we aimed at measuring treatment success by assessing different stages of OS progression, especially metastatic disease. To show clinical relevance, we performed immunofluorescence (IF) of EDA on primary human OS samples. In a second step, we evaluated drug efficacy in a syngeneic OS mouse model. K7M2L2 OS cells, stably infected to express the marker proteins lacZ and mCherry, were orthotopically injected into the tibia of mice. Once primary tumors were detectable, the mice were treated with either PBS or F8-TNF-α. The compound was given either systemically (intravenously (i.v.), tail vein) or locally (intraarterial (i.a.), femoral artery). Four days after the last treatment, all mice were sacrificed, terminal blood sampling was performed and blood samples were analyzed for the presence of circulating tumor cells (CTCs) via FACS/mCherry fluorescence. Following X-gal staining, metastases on the surface of lungs were counted. EDA and infiltration of immune cells (CD4+, CD8+, natural killer (NK) cells, CD19+) were assessed histologically. Using IF, we detected strong expression of EDA in primary tumors as well as bone and lung metastases from human OS patients. Preliminary results from our treatment study showed a significant decrease in the number of lung metastases as well as the number of CTCs in mice treated with i.a. F8-TNF-α while the treatment was well tolerated by the mice. The reduction of lung metastases could, at least in part, be explained by a strong expression of EDA in the lung metastases. In addition, significant increases in lung-infiltrating immune cells were observed after administration of F8-TNF-α (i.v. F8- TNF-α: increased CD4+ T-cells; i.a. F8-TNF-α: increased NK cells). Although significant reductions in numbers of metastases and CTCs were detected, F8-TNF-α did not significantly impair the growth of the primary tumors. In conclusion, our study demonstrates a reduction of CTCs as well as lung metastases after administration of F8-TNF-α and thus, provides a rationale for further studying the anti-metastatic effects of this compound in a clinically relevant model of OS. Citation Format: Bernhard Robl, Sander Martijn Botter, Aleksandar Boro, Dario Neri, Bruno Fuchs. Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 589.

Nivolumab (nivo) in patients (pts) with advanced (adv) NSCLC and central nervous system (CNS) metastases (mets).

9038 Background: CNS mets occur in 20%–40% of pts with adv NSCLC and are associated with poor overall survival (OS; median ≈7 mo). We evaluated nivo in this subgroup by: 1) pooling nivo-treated pts with adv NSCLC and pretreated (pretx) stable CNS mets at baseline (BL) across CheckMate 063 (phase [ph] II), 017 (ph III), and 057 (ph III); and 2) comparing OS with nivo vs docetaxel (doc) in pts with stable BL CNS mets in CheckMate 017 and 057. Methods: 1) Nivo-treated pts with adv NSCLC and pretx BL CNS mets from CheckMate 063 (n = 3), 017 (n = 9), and 057 (n = 34) were pooled to assess BL characteristics, safety, and CNS progression. 2) OS was analyzed in pts with pretx BL CNS mets and squamous (SQ; CheckMate 017) or non-SQ (NSQ; CheckMate 057) NSCLC treated with nivo 3 mg/kg Q2W vs doc 75 mg/m2 Q3W. Results: Of 46 nivo-assigned pts with pretx CNS mets, 74% had prior CNS-site radiotherapy and 85% had ≥ 2 extra-CNS sites of mets. Median follow-up was 8.4 mo (range: 0.3, 23.4); median treatment duration (n = 45; 1 pt not treated) was 2.3 mo (range: 0.03, 23.3). Any grade (gr) treatment-related (TR) adverse events (AEs) occurred in 67% of pts; gr 3–4 TRAEs occurred in 7%, with no TR deaths. CNS TRAEs occurred in 5 pts (11%) and were all gr 1–2 (paresthesia, n = 2; dizziness, somnolence, and tremor, n = 1 each). At time of overall disease progression (PD) or last tumor assessment, 33% of pts had no evidence of CNS progression (stable/decreased CNS lesions) and 52% had unequivocal progression in the CNS (15% had no post-BL CNS assessment). In pts with pretx CNS mets from CheckMate 017, median OS (events, n; HR) with nivo (n = 9) vs doc (n = 8) was 4.99 mo vs 3.86 mo (6 vs 8; not determined); in CheckMate 057, median OS with nivo (n = 34) vs doc (n = 34) was 7.61 mo vs 7.33 mo (30 vs 27; 1.04, 95% CI: 0.62, 1.76). Conclusions: Nivo was well-tolerated in pts with adv NSCLC and pretx CNS mets, with generally low-grade toxicities. One-third of pts had no evidence of CNS progression at time of PD/last assessment. In pts with SQ and NSQ NSCLC with pretx CNS mets, median OS was similar with nivo vs doc. Additional results (including OS and CNS progression rates in pts with/without pretx CNS mets and safety/efficacy of nivo in pts with untreated BL CNS mets from CheckMate 012) will be presented. Clinical trial information: NCT01721759; NCT01642004; NCT01673867.

Abstract C3: Candidate osteosarcoma metastatic drivers identified in highly clonal metastases using transposon-mediated insertional mutagenesis

Abstract Osteosarcoma (OS) is the most common primary bone tumor and the third leading pediatric cancer. Despite combined treatments, over 30% of patients with localized OS and 75% of patients with metastases at presentation experience treatment failure within 5 years of diagnosis. Identification of OS initiation, progression, and metastatic driver genes and pathways has been difficult due to the genomic instability and subsequent complexity of OS. To address this, we created transgenic mice that develop OS via Sleeping Beauty transposon-mediated mutagenesis. Using a Trp53 pathway deficient background (Trp53R270H/+) to accelerate tumorigenesis and metastasis, the T2/Onc transposon system is activated in osteoblasts via an osteoblast specific osterix- cre recombinase transgene (Osx-Cre). Our quadruple transgenic mice (Osx-Cre; R26-LSL-SB11; LSL-Trp53R270H; T2/Onc) develop OS with an average latency of 10.5 months and a penetrance of 75% (n=96), as compared to 60% (n=49) in Trp53R270H/+ controls. Importantly, the SB-accelerated OS predilection sites are more similar to human OS as compared to other models; &amp;gt;40% arose in the hind or front legs, as most human OS is appendicular in origin. SB tumors also resemble human OS in gross anatomy, histological appearance, and presence of collagen. Over 100 metastatic nodules were collected from 16 quadruple transgenic mice with OS. Analysis of recurrent SB integration sites revealed the well-known OS genes RB1 and CMYC, validating the screen, and novel genes not previously reported, including 10 genes common among metastatic nodules. Non-supervised hierarchical clustering analysis was performed on metastatic and primary tumor sets using transposon integration sites as molecular tags. Metastases from the same mice were found to be clonal derivatives from the primary tumor and generally more related to each other than to the primary tumor, even if they were collected from different tissues. Additionally, in each set of metastases from a given mouse, there was a set of transposon insertion mutations found only in the metastatic cells and not in the primary tumor. PCR confirmation experiments of metastasis-specific insertions are underway. Thus far, it is clear that some mutations identified in only metastatic nodules are present in the indicated samples and not detectable in the primary OS tumors from the same mouse. Functional validation using published in vitro assays for migration and invasion are being performed on cell lines derived from several lung and primary tumor pairs collected from the quadruple transgenic SB mice and in well-characterized human and murine OS cell lines: U2OS, HOS, MNNG/HOS, 143B, K12, and K7M2. Gene expression will be increased using stably integrated, over expression vectors containing cDNA and/ or silenced using transcription activator-like effector nuclease (TALENs) mediated gene knockout. The SB transposon screen has revealed high clonality among metastases and identified several candidate metastatic genes for further study. Citation Format: Tracy A. Powell, Branden S. Moriarity, Aaron L. Sarver, David A. Largaespada. Candidate osteosarcoma metastatic drivers identified in highly clonal metastases using transposon-mediated insertional mutagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C3.

Abstract LB-366: Everolimus (EV) potentiates Sorafenib (SOR)activity in osteosarcoma (OS) preclinical models: a combination targeting the crosstalk between ERK1/2 and mTORC1/2 signaling pathways

Abstract OS is the most frequent primary bone tumor in children and young adults.The mammalian Target of Rapamycin (mTOR) and the Extracellular Regulated Kinases (ERK)1/2 pathways have been shown to cooperate in survival advantage of OS. Inhibition of this signaling can be a rational pharmacological strategy to impinge on OS progression. This study pursued the dual blockage of ERK1/2 and mTOR pathways by combining the multikinase inhibitor SOR with the rapamycin analog EV. The synergistic anti-profilerative effect was found against 5 out 7 OS cell lines (combination index&amp;lt;1, based on Chou-Talalay method). The inhibition of OS colony growth, due to cell cycle arrest and apoptosis induction was obtained. Mechanistically SOR alone, but EV, inhibited phospho-ERK1/2. EV and SOR as single agent induced the down-regulation of P-S6, P-4EBP. EV alone induced an increase in phospho-AKT as a consequence of the release of negative feed back by MTORC1 on MTORC2. Nonetheless, the combined treatment completely abrogated both ERK1/2 and MTORC1/2. The crosstalk among these pathways was interrupted by AMPK activation due to sorafenib-induced ROS burst and MTORC2 disassembling. The anti-tumoral activity and the inhibition of angiogenesis and metastasis in vivo were demonstrated against OS xenografts in NOD/SCID mice and in chicken chorioallantoic membrane model. A 28 daily oral gavage of mice (6/group) with SOR 5mg/kg, EV 1mg/kg, or their combination reduced tumor growth to 34%, 46%, and 29% of control slowing down cell proliferation and inducing apoptosis. In vivo model of OS metastasis obtained by e.v injection of OS cells confirmed the high metastatic potential of human OS consistently with the clinical experience. However, daily treatment of mice induced a significant reduction in number and size of lung metastases (foci area 65%, 74% and 29% of control obtained after 14 daily SOR, EV, and combined treatment). Anti-migratory effect was evidenced in 7 OS cell lines and was attributable to SRC/FAK/ERM pathway inhibition. HUVEC branching morphogenesis inhibition and reduction of blood vessel density in xenografts confirmed a strong potentiation of anti-angiogenic activity of drug combination. These results straighten our understanding of the networking nature of oncogenic signal transduction circuits in OS, therefore sustaining the rational for testing combination of sorafenib and everolimus in clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-366. doi:1538-7445.AM2012-LB-366

What is the clinical benefit of preoperative chemoradiotherapy with 5FU/leucovorin for T3-4 rectal cancer in a pooled analysis of EORTC 22921 and FFCD 9203 trials: Surrogacy in question?

BackgroundTwo phase III trials of neoadjuvant treatment in T3-4 rectal cancer established that adding chemotherapy (CRT) to radiotherapy (RT) improves pathological complete response (pCR) and local control (LC). We combined trials to assess the clinical benefit of CRT on overall (OS) and progression free survival (PFS) and to explore the surrogacy of pCR and LC. Patients and methodsIndividual patient data from European Organisation for Research and Treatment of Cancer (EORTC) 22921 (1011 patients) and FFCD 9203 (756 patients) were pooled. Meta-analysis methodology was used to compare neoadjuvant CRT to RT for OS, PFS LC and distant progression (DP). Weighted linear regression was used to estimate trial-level association (surrogacy R2) between treatment effects on candidate surrogate (pCR, LC, DP) and OS. ResultsThe median follow-up was 5.6years. Compared to RT (881pts), CRT (886pts) did not prolong OS, DP or PFS. The 5-y OS-rate was 66.3% with CRT versus 65.9% in RT (hazard ratios (HR)=1.04 {0.88–1.21}). CRT significantly improved LC (HR=0.54, 95%confidence interval (CI): 0.41–0.72). PFS was validated as surrogate for OS with R2=0.88. Neoadjuvant treatment effects on LC (R2=0.17) or DP (R2=0.31) did not predict effects on OS. ConclusionPreoperative CRT does not prolong OS or PFS. pCR or LC do not qualify as surrogate for PFS or OS while PFS is surrogate. Phase III trials should use OS or PFS as primary endpoint.

Abstract 5340: Regulation of osteoblast differentiation: The pivotal role of CD99 molecule

Abstract Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. It displays a complex karyotype and a broad range of molecular abnormalities; however most of these alterations are not constantly detected in the majority of osteosarcoma tumors. Although its precise origin still remains unknown, increasing findings suggest OS should be regarded as a differentiation disease caused by genetic and epigenetic changes which interrupt osteoblast differentiation from mesenchymal stem cells (MSCs). Few sporadic data link CD99 molecule to osteoblast functions. It is a 32 kDa surface glycoprotein involved in several biological processes. We have previously demonstrated that CD99 acts as a tumor suppressor in osteosarcoma where it is either undetectable or present at very low levels. Being expressed in osteoblasts (OBs), the normal counterpart, we tested the hypothesis that CD99 may contribute to osteoblastic differentiation. Its role was assessed, first of all, in physiological conditions. We provided evidence that CD99 is present in MSCs, considered the cells of origin for OS, and its expression increases during osteoblastogenesis and osteoblast maturation evaluated in MSCs as well as in OBs. To study the function of CD99 in OS we took advantage of two osteosarcoma cell lines, Saos-2 and OS7, and their respective clones overexpressing the glycoprotein. Grown in osteogenic conditions for 21 days, these cells were able to differentiate and mineralize, as underlined by typical genes (collagen I, alkaline phosphatase and osteocalcin) as well as by specific stainings for matrix mineralization. In presence of the molecule, cells were able to restore a normal phenotype, following the normal osteogenic program, thus allowing transition toward the latter step of differentiation. An effective proliferation arrest was reached in association with withdrawal from cell cycle in G1 phase, reduction of cyclin A, high expression of p21, and massive apoptosis induction. The strong and prolonged activation of ERK in presence of CD99 suggested the involvement of MAP kinase in differentiation, as it was detected in MSCs under differentiating conditions. After PD98059 treatment, ERK inhibition really attenuated osteoblastic features in CD99 expressing cells. In conclusion, these results give new insight into the role of the glycoprotein in osteoblastic differentiation. Its forced expression helps to overcome the impaired osteoblastogenesis that occurs in OS cells. These data can open new avenue for the understanding of mechanisms regulating OS pathogenesis and progression and they also suggest a possible new therapeutic approach to OS based on modulating tumor differentiation (grant from Italian Association for Cancer Research). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5340. doi:10.1158/1538-7445.AM2011-5340