Modeling the MET Oncogene in Osteosarcoma

骨肉瘤中 MET 癌基因的建模

• 批准号: 8554717
• 负责人: Su-Young Kim
• 金额: $ 0.56万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554717
• 关键词: Affect; Aftercare; Antibodies; Attenuated; Biological; Biological Assay; Canis familiaris; Carcinoma; Cell Line; Cell Surface Receptors; Cells; Chemicals; Child; Childhood; Clinic; Clinical; Clinical Trials; Complement; Disease; Genetic; Goals; Hematologic Neoplasms; Hepatocyte Growth Factor; Hour; Human; Immunodeficient Mouse; Immunohistochemistry; In Vitro; Indium; Infection; Journals; Laboratories; Ligands; Localized Disease; Lung; Lung nodule; MET Oncogene; Malignant Bone Neoplasm; Manuscripts; Mediator of activation protein; Migration Assay; Modeling; Mus; Mutate; Mutation; Neoplasm Metastasis; Nitrosoguanidines; Nodule; Oncogenes; Operative Surgical Procedures; Outcome; Patients; Phenotype; Primary Neoplasm; Protein Tyrosine Kinase; RNA; Regimen; Role; Sampling; Survival Rate; Testing; Transformed Cell Line; Translating; Translations; Tumor Cell Line; United States; anticancer research; cell motility; chemotherapy; improved; inhibitor/antagonist; lentiviral-mediated; matrigel; meetings; member; migration; oncology; osteosarcoma; overexpression; receptor binding; research study; sarcoma; small molecule; soft tissue; tumor; urologic

BackgroundOsteosarcoma is the most common pediatric bone cancer in the United States. Advances in surgery and chemotherapy have resulted in a cure rate of 65% for children who present with localized disease. However, patients who have metastatic disease have only a 25% survival rate. Attempts to improve this outcome using various intensification regimens have not improved survival. Unfortunately, these results demonstrate the inability to target the tumor using intensification of chemotherapy to salvage patients with metastatic disease. Therefore, we have turned our efforts to identifying biological elements in this disorder that are amenable to modulation.HOS is a human osteosarcoma cell line that does not form primary tumors and does not form metastatic lung nodules in immunodeficient mice. However, treatment of HOS cells with the chemical N-methyl-N-nitro-N-nitrosoguanidine (MNNG) resulted in a transformed cell line, MNNG-HOS, that is able to form primary tumors and also metastatic nodules in the lung. Further characterization of the chemically transformed cell line resulted in the identification of the Met oncogene as the likely mediator of metastasis.Met is a cell surface receptor that binds a single ligand, hepatocyte growth factor, and has tyrosine kinase activity. Met is mutated or over-expressed in a variety of tumors, including many carcinomas and soft tissue sarcomas. In addition, a study in large dogs with osteosarcoma has shown that 10% of these tumors harbor mutations in Met. These findings led us to study Met and its possible role in osteosarcoma metastasis.We showed that Met is highly expressed in patient samples by immunohistochemistry. We also collaborated with members of Donald Bottaros laboratory in the Urological Oncology Branch to optimize a dual antibody electrochemiluminescence (ECL) assay to obtain quantitative values of expression, to show that Met was highly expressed in the vast majority of osteosarcoma cell lines and tumor samples, with a subset also expressing phosphorylated, activated Met. We went on to test various osteosarcoma cell lines to determine if they were responsive to a Met inhibitor, PHA-665752. ECL assays demonstrated significantly lower levels of phospho-Met levels in cells treated with PHA-665752. These levels returned to their high baseline levels when examined 96 hours after treatment, demonstrating that the inhibitor was targeting Met. We then tested the ability of PHA-665752 to inhibit the following parameters: proliferation (MTT analysis), motility (scratch assay), migration (through an 8 micron pore) and invasion (migration through a matrigel matrix). PHA-665752 had no effect on proliferation in any of the osteosarcoma cell lines tested. However, it was effective in inhibiting one or more of the other parameters in three osteosarcoma cell lines. These included LR, MG63.2 and MNNG-HOS. LR and MG63.2 express the highest levels of phospho-Met and MNNG-HOS has activated Met due to a translocation. These results demonstrated that only cells that have activated Met have a decrease in the metastatic phenotype in vitro when Met is inhibited.We then utilized a genetic approach to complement the pharmacological experiments. Specifically, we utilized Met specific sh-RNA constructs to demonstrate that lentiviral mediated infection of these constructs into high Met expressing osteosarcoma cell lines resulted in inhibition of in vitro parameters of metastases. We proceeded to test the above constructs in a murine model of metastases and demonstrated that mice injected with sh-MET infected osteosarcoma cell lines had prolonged survival compared to control mice. With the plan to translate these findings for clinical use, we then obtained an orally available small molecule Met specific inhibitor, PF-04217903. This compound is currently in clinical trials. Utilization of this compound in osteosarcoma cell lines affected parameters of metastasis in vitro. Inaddition, treatment of mice with this compound dramatically reduced the number of resulting metastatic lung nodules.Plans for 2012In 2012, we are preparing a manuscript detailing the above findings for submission to the journal Cancer Research. We would also like to identify the mechanism by which Met is activated in osteosarcoma.Significance and GoalsThe successful completion of the above objectives may reveal the mechanism, or mechanisms, of action by which Met is activated in osteosarcoma cell lines. Several Met-specific inhibitors are currently in clinical trials, which allows for rapid translation into the clinic, if we are able to successfully complete our objectives.

背景骨肉瘤是美国最常见的儿童骨癌。 手术和化疗的进步使患有局部疾病的儿童的治愈率达到 65%。 然而，患有转移性疾病的患者的生存率仅为 25%。 使用各种强化治疗方案来改善这一结果的尝试并未改善生存率。 不幸的是，这些结果表明无法通过强化化疗来靶向肿瘤来挽救患有转移性疾病的患者。 因此，我们将努力转向识别这种疾病中易于调节的生物元件。HOS是一种人骨肉瘤细胞系，在免疫缺陷小鼠中不会形成原发性肿瘤，也不会形成转移性肺结节。 然而，用化学物质 N-甲基-N-硝基-N-亚硝基胍 (MNNG) 处理 HOS 细胞会产生转化的细胞系 MNNG-HOS，该细胞能够形成原发性肿瘤以及肺部的转移性结节。 对化学转化细胞系的进一步表征导致 Met 癌基因被鉴定为可能的转移介质。Met 是一种细胞表面受体，可结合单一配体、肝细胞生长因子，并具有酪氨酸激酶活性。 Met 在多种肿瘤中发生突变或过度表达，包括许多癌和软组织肉瘤。 此外，一项针对患有骨肉瘤的大型犬的研究表明，这些肿瘤中有 10% 存在 Met 突变。 这些发现促使我们研究 Met 及其在骨肉瘤转移中的可能作用。我们通过免疫组织化学表明 Met 在患者样本中高表达。 我们还与泌尿肿瘤科Donald Bottaros实验室的成员合作，优化了双抗体电化学发光（ECL）测定，以获得表达的定量值，以表明Met在绝大多数骨肉瘤细胞系和肿瘤样本中高表达，其中一个子集也表达磷酸化、活化的 Met。 我们继续测试了各种骨肉瘤细胞系，以确定它们是否对 Met 抑制剂 PHA-665752 有反应。 ECL 检测表明，用 PHA-665752 处理的细胞中磷酸-Met 水平显着降低。 治疗后 96 小时检查时，这些水平恢复到较高的基线水平，表明该抑制剂以 Met 为目标。 然后，我们测试了 PHA-665752 抑制以下参数的能力：增殖（MTT 分析）、运动性（划痕测定）、迁移（通过 8 微米孔）和侵袭（通过基质胶基质迁移）。 PHA-665752 对任何测试的骨肉瘤细胞系的增殖均没有影响。 然而，它可以有效抑制三种骨肉瘤细胞系中的一种或多种其他参数。 其中包括 LR、MG63.2 和 MNNG-HOS。 LR 和 MG63.2 表达最高水平的磷酸 Met，并且 MNNG-HOS 由于易位而激活了 Met。 这些结果表明，当 Met 被抑制时，只有激活 Met 的细胞在体外的转移表型才会降低。然后，我们利用遗传方法来补充药理学实验。 具体来说，我们利用Met特异性sh-RNA构建体来证明慢病毒介导的这些构建体感染高Met表达骨肉瘤细胞系导致体外转移参数的抑制。 我们继续在小鼠转移模型中测试上述构建体，并证明注射 sh-MET 感染的骨肉瘤细胞系的小鼠与对照小鼠相比具有更长的生存期。 为了将这些发现转化为临床应用，我们获得了一种口服小分子 Met 特异性抑制剂 PF-04217903。 该化合物目前正处于临床试验阶段。 在骨肉瘤细胞系中使用这种化合物会影响体外转移的参数。 此外，用这种化合物治疗小鼠可显着减少由此产生的转移性肺结节的数量。 2012 年计划 2012 年，我们正在准备一份详细介绍上述发现的手稿，以提交给《癌症研究》杂志。 我们还想确定 Met 在骨肉瘤中激活的机制。意义和目标上述目标的成功完成可能会揭示 Met 在骨肉瘤细胞系中激活的作用机制。 几种 Met 特异性抑制剂目前正在进行临床试验，如果我们能够成功完成我们的目标，就可以快速转化为临床。