Targeting the TGIF/Twist1 network in osteosarcoma

总结 骨肉瘤是最常见的原发性骨恶性肿瘤，也是与癌症相关的第二大原因。 青少年死亡。在诊断时无明显转移的患者中， 辅助化疗的长期存活率接近70%。然而，对于患有 转移性或复发性骨肉瘤，目前没有可靠的治疗选择提供长期的肿瘤 控制，强调迫切需要更好地了解这种疾病。 在我们的努力，以解开一个潜在的作用，同源结构域蛋白TGIF在骨肉瘤发病机制 或进展，我们采用睡美人（SB）系统进行体内诱变方法。我们 发现Tgif基因的同时缺失和SB突变的激活， 间充质细胞或定向成骨细胞在高转移性骨肉瘤的发展中达到顶峰。 骨肉瘤显示p53和p16 Ink 4a（p16）的复发性丢失，这两种抑制基因在骨肉瘤中经常发生改变， 人骨肉瘤相应地，TGIF的异位表达抑制了骨肉瘤细胞的增殖 以依赖于p53和p16的方式。为了进一步分析TGIF和p53/p16之间的功能相互作用， 我们探索了我们在酵母双杂交筛选中鉴定的TGIF和Twist 1之间的物理相互作用， 因为Twist 1已被证明限制p53和p16的表达，更重要的是，Twist 1基因是 在人骨肉瘤中经常扩增或过表达。我们发现TGIF与 抑制Twist 1的转录活性，导致p53和p16的积累和随之而来的抑制， 骨肉瘤细胞增殖。在人骨肉瘤中，TGIF表达的缺失，部分是由于 TGIF基因的破坏与肿瘤的侵袭性有关，暗示TGIF是一种潜在的肿瘤抑制因子。 预后标志物和可能的目标衰减失调细胞增殖在这种恶性肿瘤。 总的来说，这些发现促使我们假设TGIF可能作为一种肿瘤抑制因子， 通过拮抗Twist 1的表达和活性，从而促进p53和 p16细胞抑制程序。我们还假设，TGIF表达的失调可能被用来定义一个新的基因。 改善骨肉瘤预后以及设计创新治疗策略 这种致命的恶性肿瘤将在以下具体目标中检验这些总体假设：目标1： 探讨TGIF在骨肉瘤发病机制和进展中的作用， 与p53、p16和Twsit 1相互作用。目的2：探讨TGIF抑制骨肉瘤的作用机制 进展，重点是其拮抗Twist 1表达和活性的能力。目标3：建立临床 骨肉瘤中TGIF表达失调的相关性全面描述这一新的 肿瘤抑制基因的发现将可能为骨肉瘤领域开辟新的机会， 描绘潜在的机制，以及预后和药物发现。

SUMMARY Osteosarcoma is the most frequent primary malignancy of bone and the second leading cause of cancer-related death in adolescents. In patients without demonstrable metastasis at the time of diagnosis, surgical resection and adjuvant chemotherapy have resulted in long-term survival rates that approach 70%. However, for patients with metastatic or relapsed osteosarcoma, there is currently no reliable therapeutic option to provide long-term tumor control, emphasizing the urgent need for a better understanding of the disease. In our efforts to unravel a potential role for the homeodomain protein TGIF in osteosarcoma pathogenesis or progression, we undertook an in vivo mutagenesis approach using the Sleeping Beauty (SB) system. We found that simultaneous deletion of the Tgif gene and activation of SB mutagenesis either in limb bud mesenchymal cells or in committed osteoblasts culminated in the development of highly metastatic osteosarcomas that display recurrent loss of p53 and p16Ink4a (p16), two suppressor genes frequently altered in human osteosarcoma. Congruently, ectopic expression of TGIF suppressed proliferation of osteosarcoma cells in a manner dependent on p53 and p16. To further dissect the functional interplay between TGIF and p53/p16, we explored a physical interaction between TGIF and Twist1 that we identified in a yeast two hybrid screening, because Twist1 has been shown to restrict both p53 and p16 expression, and more crucially, the Twist1 gene is frequently amplified or overexpressed in human osteosarcoma. We found that TGIF associated with and repressed Twist1 transcriptional activity, leading to p53 and p16 accumulation and an attendant suppression of osteosarcoma cell proliferation. In human osteosarcoma, loss of TGIF expression, which is due in part to the disruption of the TGIF gene, is associated with tumor aggressiveness, implicating TGIF as a potential prognostic marker and possible target for attenuating deregulated cell proliferation in this malignancy. Collectively, these findings prompted us to hypothesize that TGIF might function as a tumor suppressor in osteosarcoma by antagonizing Twist1 expression and activity and thereby facilitating integration of the p53 and p16 cytostatic programs. We also hypothesize that deregulated TGIF expression might be exploited to define a signature to improve prognostic of osteosarcoma as well as to design innovative therapeutic strategies to curb this lethal malignancy. These overarching hypotheses will be tested in the following specific aims: Aim 1: Investigate the role of TGIF in osteosarcoma pathogenesis and progression, focusing on its functional interaction with p53, p16, and Twsit1. Aim 2: Explore the mechanisms by which TGIF restrains osteosarcoma progression, focusing on its ability to antagonize Twist1 expression and activity. Aim 3: Establish the clinical relevance of deregulated TGIF expression in osteosarcoma. Comprehensive characterization of this newly discovered tumor suppressor gene will likely open up new opportunities to the osteosarcoma field, both in terms of delineating the underlying mechanisms, and in terms of prognosis and drug discovery.