Targeting the TGIF/Twist1 network in osteosarcoma

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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.