Activity In Osteosarcoma Research Articles

Restoration of microRNA-130b expression suppresses osteosarcoma cell malignant behavior in vitro.

The alteration of microRNA (miR)-130b expression has been associated with promoting or suppressing numerous types of human cancer. A previous study evaluated the expression level of miR-130b in osteosarcoma tissues, and subsequently investigated the effects of miR-130b on the regulation of osteosarcoma cells malignant behavior in vitro. The study revealed that miR-130b expression levels were significantly reduced in osteosarcoma tissues and cell lines, compared with in adjacent tissues or normal cell lines. The expression of miR-130b inhibited the proliferation of osteosarcoma U-2OS and Saos-2 cells and impaired their ability to migrate, invade and form colonies. Furthermore, analysis using TargetScan and a dual-luciferase reporter assay demonstrated that miR-130b directly interacted with the 3′-untranslated region of transforming growth factor α (TGFA) and suppressed TGFA expression. TGFA and miR-130b were also inversely expressed in osteosarcoma tissues. In addition, expression of TGFA was able to alter miR-130-regulated osteosarcoma cell proliferation, migration and invasion. Thus, the present study demonstrated that miR-130b was downregulated in osteosarcoma tissues and cell lines, whereas the expression of miR-130b suppressed osteosarcoma cell malignant behavior. At the gene level, miR-130 directly targets and inhibits TGFA expression, in addition to phosphorylated protein kinase B and epidermal growth factor receptor expression levels. Further study is required to evaluate miR-130b antitumor activity in osteosarcoma.

Aurora B Kinase as a Novel Molecular Approach for Osteosarcoma Therapy

Osteosarcoma is the primary human malignant tumor affecting bone. This cancer most frequently arises in children and adolescents, with a second peak in those over the age of 50. Currently, surgery followed by radiotherapy and chemotherapy are the main treatments, but long-term positive effects are very poor. Aurora B kinase is a serine/threonine kinase that is a key regulator of cell cycle and mitosis. Tissue array analysis revealed that Aurora B kinase is overexpressed in osteosarcoma compared with normal bone tissue. We developed a compound, HOI-07 [i.e., (E)-3-((E)-4-(benzo[d] [1,3]dioxol-5-yl)-2-oxobut-3- en-1-ylidene)indolin-2-one], as a specific Aurora B kinase inhibitor and examined its effectiveness against osteosarcoma cell growth in this study. This compound inhibited Aurora B kinase activity in osteosarcoma and induced apoptosis, caused G2-M phase arrest, and attenuated osteosarcoma anchorage-independent cell growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of osteosarcoma to HOI-07. Results of a xenograft mouse study indicated that HOI-07 treatment effectively suppressed the growth of 143B and KHOS xenografts, without affecting the body weight of mice. The expression of phosphorylated histone H3 (Ser10) was reduced in mice treated with HOI-07. Overall, we identified HOI-07 as a specific Aurora B kinase inhibitor for osteosarcoma treatment and this compound warrants further investigation. Funding Statement: This work was funded by The Hormel Foundation and National Institutes of Health grants CA166011, CA187027, and CA196639 and a Joint Funds of the National Natural Science Foundation of China (Grant No. 162300410337) Declaration of Interests: The authors declare none. Ethics Approval Statement: Mice were maintained under “specific pathogen-free” conditions based on the guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee. PDX animal studies were preformed following guidelines approved by the Zhengzhou University Institutional Animal Care and Use Committee (Zhengzhou, Henan, China).

Abstract 186: FDA approved drug Bazedoxifene as a novel inhibitor of IL 6 and IL 11/GP130 signaling for osteosarcoma therapy

Abstract IL-6 and L-11 signaling pathways play an important role in cancer cell survival and progression, including osteosarcoma. IL-6/IL-11 binds to IL-6/IL-11 Rα to form a binary complex, then recruits GP130 to form the IL-6/IL-6 Rα/GP130 or IL-11/IL-11 Rα/GP130 heterotrimer and triggers a signaling cascade downstream. One of the major downstream effectors of IL-6 is STAT3. STAT3 is persistently activated in many human osteosarcoma specimens and cell lines and STAT3 is required for osteosarcoma cells survival. So IL-6 and IL-11 present a viable novel target for osteosarcoma therapy. To date, however, no small molecules that target both IL-6/GP130 and IL-11/GP130 signaling pathways are available for cancer therapy. To overcome this critical problem, we have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repositioning to target GP130. Drug repositioning refers to reuse fragments from the existing FDA-approved drugs for new applications and could potentially reposition the existing drugs as novel, off-target inhibitors of the GP130 as their new target. Using this novel method, we have identified a FDA-approved drug Bazedoxifene with a novel function to inhibit IL-6 and GP130 proteinprotein interactions. Furthmore, because IL-11 binds to the same domain of GP130 as IL-6, Bazedoxifene should also inhibit IL-11/GP130 signaling. Accordingly, Bazedoxifene can indeed inhibit the induction of P-STAT3 by both IL-6 and IL-11. Bazedoxifene appeared specific to IL-6 and IL-11 as phosphorylation of STAT1 and STAT3 by IFN-γ and LIF respectively were not affected by the compound. Bazedoxifene inhibited P-STAT3 and induced apoptosis in human osteosarcoma cell lines expressing IL-6 and IL-11. In addition, Bazedoxifene can inhibit colony formation after the drug treatments in cancer cells.IL-6 but not IFN-γ could rescue the Bazedoxifene-mediated inhibition of cell viability in osteosarcoma cells. These results further support that IL-6/GP130 signaling pathway is one of the main targets of Bazedoxifene-mediated inhibition in osteosarcoma cells. To determine the in vivo activity of Bazedoxifene, we further tested the efficacy of Bazedoxifene in tumor xenografts generated from the SJSA osteosarcoma cells that show persistent IL-6/STAT3 activation. Bazedoxifene via oral gavage inhibited P-STAT3 and the growth of SJSA tumor xenografts. These data demonstrated that Bazedoxifene is potent in suppressing tumor growth and is orally bioavailable in inhibiting P-STAT3. It further indicates that Bazedoxifene is a promising IL-6/GP130-targeting drug, which likely to have in vivo anti-tumor activity in osteosarcoma. In summary, Bazedoxifene already approved for safety by the FDA as a novel inhibitor of IL-6/GP130 and IL-11/GP130 signaling should provide an easier path to clinical trials and have a potential to improve the outcome of osteosarcoma. Note: This abstract was not presented at the meeting. Citation Format: Xiaojuan Wu, Hui Xiao, Chenglong Li, Jiayuh Lin. FDA approved drug Bazedoxifene as a novel inhibitor of IL 6 and IL 11/GP130 signaling for osteosarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 186. doi:10.1158/1538-7445.AM2017-186

The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/βIII-Tubulin Axis.

MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintains normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma; however, the molecular mechanisms underlying miR-34a activity in osteosarcoma are not well-defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in osteosarcoma. Levels of miR-34a were attenuated in human osteosarcoma cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pull-down assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3'-untranslated region. Overexpression of miR-34a in osteosarcoma cells suppressed STMN1 expression and reduced cell growth in vitro Restoration of miR-34a led to microtubule destabilization and increased βIII-tubulin expression, with corresponding G1-G2 phase cell-cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated βIII-tubulin expression in osteosarcoma cells, suggesting that miR-34a indirectly affects Sp1. Validating the coordinating role of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in osteosarcoma cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates βIII-tubulin, leading to disruption of the microtubule network and cell death.Implications: The miR-34a/STMN1/βIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy. Mol Cancer Res; 15(7); 953-64. ©2017 AACR.

BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

H19 Functions as a ceRNA in Promoting Metastasis Through Decreasing miR-200s Activity in Osteosarcoma.

Osteosarcoma is the most common primary bone sarcoma around the world. The poor prognosis and high recurrence rate of osteosarcoma are largely due to the high rate of pulmonary metastasis. H19 has been reported to play a potential role in osteosarcoma progression. However, the exact molecular mechanism of metastasis involving H19 remains unclear. In the present study, we performed gain- and loss-of-function assays and found that H19 promotes migration and invasion in osteosarcoma cells. Furthermore, we showed that H19 promotes metastasis through upregulation of ZEB1 and ZEB2 by competitively binding the miR-200 family. Thus, our findings suggest important roles of H19 in osteosarcoma metastasis and indicate its potential application in osteosarcoma therapy.

IGF1 3'UTR functions as a ceRNA in promoting angiogenesis by sponging miR-29 family in osteosarcoma.

Osteosarcoma is one of the most common malignant bone tumors in human worldwide. Angiogenesis is a pivotal process during osteosarcoma development. Insulin-like growth factor 1 (IGF1) has been reported to promote angiogenesis. However, the role of 3' untranslational region (3'UTR) of IGF1 mRNA in angiogenic activity in osteosarcomas is still unknown. In the present study, we performed gain-of-function assays to investigate the role of IGF1-3'UTR in angiogenesis. For the first time, we demonstrated that IGF1 3'UTR increased VEGF expression and promotes angiogenesis in osteosarcoma cells. In addition, RNA-immunoprecipitation and luciferase reporter assays showed that IGF1 3'UTR was a direct target of miR-29s. Our data also demonstrated that there existed a competition of miR-29s between IGF1-3'UTR and VEGF mRNA, and IGF1-3'UTR promoted angiogenesis at least in part via sponging miR-29s. Taken together, our study suggests that IGF1-3'UTR functions as a ceRNA in promoting angiogenesis by sponging miR-29s in osteosarcoma.

Investigation of osteosarcoma genomics and its impact on targeted therapy: an international collaboration to conquer human osteosarcoma

Osteosarcoma is a genetically unstable malignancy that most frequently occurs in children and young adults. The lack of progress in managing this devastating disease in the clinic has prompted international researchers to collaborate to profile key genomic alterations that define osteosarcoma. A team of researchers and clinicians from China, Finland, and the United States investigated human osteosarcoma by integrating transcriptome sequencing (RNA-seq), high-density genome-wide array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, cell culture, and molecular biological approaches. Systematic analysis of genetic/genomic alterations and further functional studies have led to several important findings, including novel rearrangement hotspots, osteosarcoma-specific LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes, VEGF and Wnt signaling pathway alterations, deletion of the WWOX gene, and amplification of the APEX1 and RUNX2 genes. Importantly, these genetic events associate significantly with pathogenesis, prognosis, progression, and therapeutic activity in osteosarcoma, suggesting their potential impact on improved managements of human osteosarcoma. This international initiative provides opportunities for developing new treatment modalities to conquer osteosarcoma.

Correction: Driven to Death: Inhibition of Farnesylation Increases Ras Activity in Osteosarcoma and Promotes Growth Arrest and Cell Death

Correction: Driven to Death: Inhibition of Farnesylation Increases Ras Activity in Osteosarcoma and Promotes Growth Arrest and Cell Death

Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma.

Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. Among new therapeutic approaches, zoledronic acid (ZOL) represents a promising adjuvant molecule to chemotherapy to limit the osteolytic component of bone tumors. However, ZOL triggers the elevation of heat shock proteins (Hsp), including Hsp27 and clusterin (CLU), which could enhance tumor cell survival and treatment resistance. We hypothesized that targeting CLU using siRNA or the antisense drug, OGX-011, will suppress treatment-induced CLU induction and enhance ZOL-induced cell death in osteosarcoma (OS) cells. The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS). In OS cell lines, ZOL increased levels of HSPs, especially CLU, in a dose- and time-dependent manner by mechanism including increased HSF1 transcription activity. The OS resistant cells to ZOL exhibited higher CLU expression level than the sensitive cells. Moreover, CLU overexpression protects OS sensitive cells to ZOL-induced cell death by modulating the MDR1 and farnesyl diphosphate synthase expression. OGX-011 suppressed treatment-induced increases in CLU and synergistically enhanced the activity of ZOL on cell growth and apoptosis. These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 20mg/kg), potentiated the effect of ZOL (s.c; 50µg/kg), significantly inhibiting tumor growth by 50% and prolonging survival in MNNG/HOS xenograft model compared to ZOL alone. These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

Gemcitabine and docetaxel for the treatment of children and adolescents with recurrent or refractory osteosarcoma: Korea Cancer Center Hospital experience

We evaluated the efficacy of gemcitabine and docetaxel chemotherapy (GEM + DOC) in children and adolescents with recurrent or refractory osteosarcoma. Data of 28 patients (20 male, 8 female) who received gemcitabine (675 or 900 mg/m(2) on days 1 and 8) and docetaxel (100 mg/m(2) on day 8) at Korea Cancer Center Hospital were retrospectively reviewed. Patients (ages 5.0-19.7 years) received a total of 96 courses of chemotherapy (median 3 courses; range, 1-8 courses) and were followed for a median of 14.9 months (range, 0.6-81.4 months). Eleven patients received GEM + DOC after surgery as adjuvant chemotherapy. Seventeen patients received GEM + DOC as palliative therapy, and were eligible for response evaluation; there were three (17.6%) complete response (CR, including two metabolic CR), one (5.9%) partial responses (PR), and three (29.4%) stable disease (SD). The objective response rate (CR + PR) and tumor control rate (CR + PR + SD) were 23.5% and 41.2%, respectively. The median duration of response was 11.2 months (range, 2.8-14.6 months). Dose of gemcitabine (675 or 900 mg/m(2)) did not influence the response rate. Overall survival at 1-year was 53.6 ± 9.4% and patients who received GEM + DOC as adjuvant chemotherapy fared better than those who received GEM + DOC as palliative therapy (72.7 ± 13.4% vs. 35.3 ± 11.6%, P = 0.006). GEM + DOC showed some activity in osteosarcoma. Better than expected survival after GEM + DOC was seen both in patients with and without surgery. These results may indicate that dose dense combinations of gemcitabine and taxanes (e.g., gemcitabine + nab-paclitaxel) should be investigated in bone sarcomas.

Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Abstract 3980: The immunomodulatory agent L-MTP-PE induces activation and expansion of human γδ T cells capable of lysing osteosarcoma cells.

Abstract The outcome of patients with metastatic osteosarcoma remains poor despite aggressive multimodality therapy. Liposomal Muramyl Tripeptide Phosphatidyl Ethanolamine (L-MTP-PE) is a non-specific immune modulator that activates monocytes and macrophages and has shown antitumor activity in osteosarcoma. The mechanism of action of L-MTP-PE remains unresolved. Here we investigated the effects of the agent on lymphocyte subpopulations. Whole peripheral blood from 9 healthy donors were stimulated with L-MTP-PE for 24 hours. Compared to non-stimulated controls, a proliferative response of CD3+ lymphocytes was found (1.77±0.06 fold expansion, p=<0.001). The strongest effect was observed on the γδ TCR+ CD3+ T cell subpopulation which expanded 5.0±1.0 fold. To further evaluate the effects of L-MTP-PE on γδ T cell proliferation, mononuclear cells were isolated from peripheral blood of 5 healthy donors and cultured in the presence of L-MTP-PE. On day 11, γδ T cells had expanded 4.6±0.6 fold compared to medium-controls. Addition of interleukin-2 (IL-2) to the cultures increased expansion of γδ T cells up to 9.8±1.6. In comparison, the known γδ T-cell stimulatory aminobisphosphonate agent zoledronic acid (ZA) combined with IL-2 induced 25.8±7.2-fold expansion. γδ T cell stimulation by ZA was previously shown to involve inhibition of the mevalonate pathway enzyme farnesyl diphosphate synthase and subsequent increase of isopentyl pyrophosphate levels. To explore the mechanism of γδ T cell expansion by L-MTP-PE, coincubation experiments were repeated in the presence of the HMG-CoA reductase inhibitor mevastatin. Addition of mevastatin resulted in a 5.1±1.8-fold reduction of L-MTP-PE induced γδ T cell expansion, confirming a critical role of the mevalonate pathway in γδ T cell expansion and thus a mechanism comparable to ZA. To explore the in vitro antitumor activity of L-MTP-PE activated γδ T cells, 24 hour coincubation experiments were performed with the osteosarcoma cell lines HOS and SAOS on day 6 of cultures. In a flow-cytometry based cytotoxicity assay, substantial cytolysis of osteosarcoma cells from both cell lines was observed after coincubation with both L-MTP-PE- and L-MTP-PE/IL-2-stimulated γδ T cells. At an effector-to-target-ratio of 1:1, L-MTP-PE-stimulated γδ T cells reduced viable cell counts of HOS and SAOS to 41.4±2.7% and 54.0±7.6%, respectively, and L-MTP-PE/IL-2-stimulated γδ T cells decreased cell counts to 33.7±2.0% and 23.0±0.9%. In comparison, ZA/IL-2-stimulated γδ T cells resulted in reduced viability to 46.2±1.0% (HOS) and 38.9±3.8% (SAOS). We conclude that L-MTP-PE induces effective in vitro expansion of γδ T cells via a mechanisms involving the mevalonate pathway, in a manner similar to aminobisphosphonate agents. L-MTP-PE-activated γδ T cells have the potential to lyse osteosarcoma cells and may thus be involved in the antitumor effects of this agents. Citation Format: Sibylle Mellinghoff, Bianca Altvater, Uta Dirksen, Heribert Juergens, Claudia Rossig, Martina Ahlmann. The immunomodulatory agent L-MTP-PE induces activation and expansion of human γδ T cells capable of lysing osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2013-3980

Abstract 4941: A microRNA activity map of human mesenchymal tumors: An integrative transcriptomic study

Abstract Background MicroRNAs (miRNAs) are nucleic acid regulators of many human mRNAs, and are associated with biological processes including cell proliferation, differentiation, and apoptosis. miRNA expression levels have been used in cancer profiling studies, but some evidence suggests that they do not fully capture miRNA regulatory activity. In this study we have integrated multiple mRNA and miRNA expression datasets to use gene expression as a surrogate for determining miRNA regulatory activity. We discovered (and validated using a paraffin-based cohort) patterns associated with cancer phenotypes and oncogenic pathways in mesenchymal tumors - a very heterogeneous class of malignancies - and we investigated possible causes for miRNA activity changes. Results Using a recently developed computational method which determines miRNA activity from changes in gene expression profiles, we identified differentially activated miRNAs between 77 normal and 135 sarcoma specimens and we validated many of these findings with microarray interrogation of an independent, paraffin-based tumor cohort of 18 samples. Among these differentially activated miRNAs are 17 with a histological subtype-specific activity pattern in comparison to normal mesenchymal tissue. We also found 81 miRNAs which exhibit a pattern common to all histological subtypes, and we identified 12 miRNAs with unique activity in osteosarcomas. In addition, we have shown that miRNA activity is imperfectly correlated with miRNA expression levels. Using next-generation miRNA sequencing we identified a specific sequence alteration which may contribute to differential activity by changing mRNA/miRNA complementarity. We then analyzed miRNA activity changes related to the RAS-pathway and found 21 miRNAs that switch from silenced to activated status in parallel with RAS activation. Importantly, nearly half of these miRNAs were predicted to regulate integral parts of the miRNA processing machinery, and our gene expression analysis revealed significant reductions of these transcripts in RAS-active tumors. This suggests an association between RAS signaling and miRNA processing in which miRNAs may attenuate their own biogenesis. Conclusions Our study represents the first gene expression-based investigation of miRNA regulatory activity in human sarcomas, and our findings indicate that miRNA activity patterns derived from integrated transcriptomic data are reproducible and biologically informative in cancer. We identified an association between RAS signaling and miRNA processing, and demonstrated a sequence alteration as a plausible cause for differential miRNA activity. Finally, our study highlights the value of systems level integrative miRNA/mRNA assessment with high-throughput genomic data, and the applicability of paraffin-tissue-derived RNA for validation of novel findings. 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 4941. doi:1538-7445.AM2012-4941

Gemcitabine and docetaxel (GEMDOX) for the treatment of relapsed and refractory pediatric sarcomas

Patients with relapsed pediatric sarcomas have a poor outcome and are in need of novel effective therapies. We retrospectively reviewed the records of patients at Children's Healthcare of Atlanta who were treated with gemcitabine (675 mg/m(2)) intravenously (IV) on Day 1 and Day 8, and docetaxel (75 mg/m(2)) IV on Day 8, repeated every 3 weeks. Nineteen patients with a median age of 11 years were treated from 2006-2010 and received 123 total courses. Two patients (11%), both with rhabdomyosarcoma, demonstrated objectives responses [one complete response (CR) and one partial response (PR)]. Seven other patients (39%) had stable disease (SD). The 1-year progression-free survival (PFS) of the entire cohort was 24% ± 10% with a median time to progression of 2 months (range: 0.5-14 months). The 1-year overall survival (OS) was 43% ± 11%. Grade 3 or 4 toxicities occurred in 14 patients (74%) and 52 courses (42%), and were most commonly hematologic (neutropenia = 37, anemia = 17, and thrombocytopenia = 23 courses). The dismal outcomes for patients with relapsed and refractory sarcomas and the lack of effective sarcoma salvage regimens highlight the need for new approaches. This report of the therapeutic activity of gemcitabine and docetaxel (GEMDOX) in rhabdomyosarcoma and other pediatric reports describing activity in osteosarcoma and Ewing sarcoma suggest that this combination should be considered for formal evaluation in a pediatric specific clinical trial. At a minimum, it appears to offer a reasonable, tolerable, palliative option.

USP1 Deubiquitinates ID Proteins to Preserve a Mesenchymal Stem Cell Program in Osteosarcoma

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.

Abstract 5283: MicroSPECT imaging of IGF1R expression with radiolabeled R1507 in osteosarcoma models

Abstract Introduction: Osteosarcoma is an aggressive form of bone cancer affecting mainly children and young adults. Despite intensive chemotherapeutic schedules, many patients still die from the disease. Therefore, new treatment strategies are being explored, such as anti-IGF1R antibody treatment. Early results from phase II studies using IGF1R antibodies demonstrated clinically relevant activity in osteosarcomas, and there is a clear need for optimal patient selection for this new treatment. First of all, membranous tumor IGF1R expression is required to benefit from this therapy. However, accurate IGF1R expression levels remain difficult to determine, since target expression can change over time and patients may present with multiple tumor manifestations demonstrating variable target expression. Obviously, it is not feasible to perform multiple biopsies. Therefore, the aim of the present study is to develop a noninvasive in vivo imaging method using radiolabeled antibodies, to visualize IGF1R expression in osteosarcomas. Methods: R1507, a monoclonal antibody directed against an extracellular epitope of the IGF1R, was radiolabeled with 111Indium (111In) and used to determine IGF1R expression in vivo in BALB/c nude mice bearing either IGF1R-positive or IGF1R-negative subcutaneous human osteosarcoma xenografts (OS1 and OS33, respectively). Mice were i.v. injected with 3 μg R1507 as optimal tumor targeting dose, or with an excess of unlabeled R1507 (300 μg) to determine the nonspecific accretion of the antibody in the tumor. Biodistribution and microSPECT imaging studies were performed 1, 3 and 7 days p.i. in mice with OS1 xenografts and at day 3 p.i. in mice with OS33 xenografts. Results: Biodistribution studies showed specific accumulation of 111In-R1507 in the OS1 xenografts from day 1 p.i. onwards (15.7 ± 4.0 %ID/g), and uptake levels increased even further on day 3 and 7 (27.5 ± 6.5 %ID/g and 25.8 ± 5.8 %ID/g, resp.). Uptake in other organs was low, and best tumor-to-blood ratios were seen at day 7 p.i. (4.5 ± 1.4). The accumulation was clearly visualized with microSPECT from day 1 p.i. onwards. In contrast, 111In-R1507 uptake in the IGF1R-negative OS33 xenografts did not exceed background levels (5.5 ± 0.6 %ID/g; tumor-to-blood: 1.2 ± 0.4), and differed significantly from the uptake in the OS1 tumors at all time points (p<0.05). On microSPECT scans, IGF1R-positive tumors could therefore clearly be distinguished from IGF1R-negative tumors. Conclusion: 111In-R1507 microSPECT imaging is an excellent method to visualize membranous IGF1R expression in vivo in human osteosarcoma xenografts. In addition, IGF1R-negative xenografts remained negative on microSPECT scans. These findings confirm that this novel technique can potentially be used to noninvasively determine IGF1R expression in osteosarcoma patients, which may allow better selection of patients who could benefit from IGF1R targeted therapy. 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 5283. doi:10.1158/1538-7445.AM2011-5283

Glycosaminoglycans as Potential Regulators of Osteoprotegerin Therapeutic Activity in Osteosarcoma

Osteosarcoma is the most frequent primary bone malignant tumor that develops mainly in children and adolescents. Despite recent improvements in chemotherapy and surgery, survival rate is approximately 50% after 5 years. Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, but its use as therapeutic agent in cancer-associated osteolysis remains controversial due to its ability to bind and inhibit the apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on tumor cells. The therapeutic effects of full-length OPG (1-401) and OPG 1-194 lacking its heparin-binding domain delivered by nonviral gene therapy were compared in a murine model of osteolytic osteosarcoma. Tumor incidence, progression, and associated bone lesions were significantly diminished in the OPG 1-194 group, but not in the OPG 1-401 group, compared with controls. As receptor activator of nuclear factor-kappaB ligand (RANKL), TRAIL, and glycosaminoglycans (GAG) were shown to be overexpressed in osteosarcoma environment compared with control tissue, OPG 1-401 bioactivity may be modulated by one of these protagonists. Surface plasmon resonance analyses performed with OPG, TRAIL, and GAGs revealed that TRAIL binds both forms of OPG with the same affinity. In addition, as OPG 1-194 and OPG 1-401 similarly inhibit TRAIL-induced apoptosis, it suggests that TRAIL is not involved in the modulation of OPG bioactivity. However, as GAGs inhibit OPG 1-401 but not OPG 1-194 binding to TRAIL or to RANKL, they may represent potent regulators of OPG availability and antitumor activity in bone tumor microenvironment.

Anti‐tumor efficacy of a transcriptional replication‐competent adenovirus, Ad‐OC‐E1a, for osteosarcoma pulmonary metastasis

Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor-specific manner are a potential modality for OSPM therapy. We detected OC mRNA expression by RNA in situ hybridization in OSA and OSPM samples from patients, and tested OC promoter transcriptional activity in OSA and non-OSA cell lines. Then we used a transcriptional replication-competent adenovirus, Ad-OC-E1a, to treat OSPM, and evaluated its tumor-specific replication and killing activities in vitro as well as anti-OSPM efficacy in vivo via systemic delivery. OC mRNA was detected in all types of OSA tissues, including OSPM tissues. The transcriptional activity of the OC promoter was much higher in a OSPM cell line SAOS-2LM7 and primary OSA cell line MG63 than in non-OSA cell lines, including cell lines from breast cancer, colon cancer, and liver cancer. Ad-OC-E1a expressed E1a protein only in MG63 and SAOS-2LM7, which indicated that adenovirus E1a was under strict control by the OC promoter. Ad-OC-E1a demonstrated killing and viral replication activity close to wild-type adenovirus levels in MG63 and SAOS-2LM7, but the killing and viral replication activities were attenuated significantly in cells expressing low OC transcriptional activity. To test whether Ad-OC-E1a could be used to target human OSPM in vivo, SAOS-2LM7 pulmonary metastasis models in nude mice were induced and treated by tail-vein injection with Ad-OC-E1a. Compared to tumor nodules in the lung in groups treated with PBS or control virus, the quantity of metastasized tumor nodules decreased significantly. Adenovirus-infected cells were stained immunohistochemically only inside and around the OSPM nodules but spared normal lung tissue and other organs. These data demonstrated that OC promoter could direct adenovirus replication by controlling the E1a gene to target human OSPM in a tumor-specific manner, providing an efficient tool to develop a feasible therapeutic modality for OSPM.

Telomerase, P53 and PCNA activity in osteosarcoma

Aims: The aim of this study was to investigate telomerase activity and to assess the correlation between telomerase activity, tumor proliferative activity and p53 overexpression in osteosarcoma tumor samples. Methods: Using a telomerase polymerase chain reaction-enzyme-linked immunoassay based on the telomeric repeat amplification protocol method, we examined telomerase activity in 26 primary osteosarcoma specimens. P53 overexpression was identified using immunohistochemical staining, and tumor proliferative activity was assessed by immunohistochemical staining of PCNA. Results: Telomerase activity was detected at a relatively low level in five of 26 osteosarcoma tissue specimens. P53 was detected in eight of 21 cases. There was no significant correlation between telomerase activity and p53 overexpression ( p=0.325). There was a significant correlation between PCNA staining and telomerase activity ( p=0.0075). Conclusion: Difference between the telomerase activity and p53 overexpression in osteosarcoma suggests that p53 and telomerase may not cooperate in tumor proliferation. Correlation of telomerase activity to PCNA expression suggests that telomerase activity may also useful for evaluate proliferative activity in osteosarcoma.