Indian hedgehog signaling in osteosarcoma: Co-expression of GLI1, PTCH1, and IHH

1010 Objectives: The Hedgehog (Hh) signaling pathway is crucial in the developmental processes of numerous tissues and organs by influencing the transcription of many target genes. Hedgehog signaling deregulation has been associated with several cancers. Indian Hedgehog (IHH) signals through the transmembrane proteins patched (PTCH) and smoothened (SMO) to activate GLI mediated transcription, including GLI1 and PTCH1. This exerts a feedback mechanism to regulate further IHH signaling. We characterized the expression of IHH, PTCH1, and GLI1 in both osteosarcoma cell lines and primary tumor specimens to determine the activity of Hedgehog signaling in osteosarcoma. The hedgehog antagonist, cyclopamine, was used to determine the proliferative role of hedgehog signaling in osteosarcoma cell lines. Methods: Fresh frozen tumor specimens were obtained from 43 patients with high grade osteosarcoma of the extremity, including 11 who presented with metastases at diagnosis (MetDx), and 32 who had no systemic disease at presentation (NoMetDx). Six osteosarcoma cell lines obtained from ATCC (U-2 OS, Saos-2, MG-63, MNNG/HOS, HOS, and KHOS/NP) were studied. RNA was extracted and real time PCR was used to assess the expression level of IHH, GLI1, and PTCH1. Satterthwaite9s t-test for unequal variance was used for tests comparing outcome groups. Non-parametric tests (Wilcoxon for binary and Jonkheere-Terpstra for continuous variables) were used to assess the association between expression levels and selected clinical and demographic variables. A bromodeoxyuridine incorporation assay was utilized to determine changes in osteosarcoma cell growth upon treatment with cyclopamine. Results: We observed a strong positive association of GLI1, IHH, and PTCH1 with each other. The pairwise correlation coefficient between GLI1 and IHH was +0.442 (p=0.003), between GLI1 and PTCH1 was +0.751 (p Conclusions: We have demonstrated that the expression levels of genes in the Hedgehog pathway are positively correlated with each other. Overexpression of these genes may result in Hedgehog pathway deregulation, which may lead to the progression of osteosarcoma. Cell culture systems are being used to further elucidate the proliferative role of this pathway in osteosarcoma. We are currently investigating the relationship between GLI1, PTCH1, IHH, and survival.