Background: The prognosis of gastric adenocarcinoma (GAC) patients with metastases is very poor. Understanding of molecular biology is limited. Hedgehog (Hh) signaling plays an important role in many tumor types and expression of Shh/Gli-1, two major molecules in Hh pathway has been documented in GAC. However, their clinical impact on GAC patients particularly in peritoneal metastasis remains elusive. Methods: Expression of Gli1 and Shh were examined using IHC in tissue microarrays containing more than 500 cases of GAC tissues with clinical annotation. The prognostic variables were determined using univariate and multivariate Cox regression analyses. GAC cell lines, patient-derived peritoneal metastatic cells and novel PDX metastatic model were used to determine the functional role of Shh/Gli-1 in vitro and in vivo. Genetic knockout Gli-1 using LentiCRISPR/Cas9 and Hh inhibitor GDC0449 as well as BET inhibitor were used to test their antitumor activities in GAC cell line and patient-derived cells. Cell proliferation, colony formation, invasion, tumor sphere assays and immunofluorescence were performed to evaluate their functionality and effects of targeted therapy. Results: Both Gli1 and Shh expression are significantly overexpressed in GAC tissue. Among 519 GAC cases, 80.76% and 87.02% were positive for nuclear Gli-1 and cytoplasmic Shh expression respectively, while the strong nuclear expression rate for Gli-1 is 69.56% and 50.10% for Shh. In the univariate Cox analysis, the overall survival was shorter for patients with high Gli-1 (p=0.018) or high Shh expression (p=0.038). In the multivariate cox analysis for both markers, only Gli-1 remained as an independently prognostic for short survival. We also observed high Gli-1 nuclear expression correlated with the presence of lymph node metastasis (p=0.032). Gli1 was highly expressed in most human malignant ascites cells. Interestingly, Gli-1 was significantly upregulated in mouse PDX-ascites cells compared to primary mice tumors. Genetic knockdown Gli-1 or pharmacologically inhibition of Gli-1 by GDC0449 Hh inhibitor or BET inhibitor JQ1 decreased Gli-1 and restored E-cadherin expression and significantly suppressed malignant cell properties and reduced population of cancer stem cells (ALDH1+ or CD133+) in patients' derived metastatic cells. Conclusions: These findings indicate that overexpression of Gli1 and Shh plays an important role in progression of peritoneal metastases of GAC. Targeting Gli1/Hh signaling may provide novel therapeutic strategies for GAC patients with peritoneal metastases. Keywords:Hh pathway, Gli1, Shh, Gastric cancer, metastasis

Citation Format: Yan Xu, Yuan Li, Bovey Liu, Melissa Pool Pizzi, Yongxi Song, Kazuto Harada, Ailing Scott, Lang Ma, Jiankang Jin, Xiaochuan Dong, Ying Wang, Brian D. Badgwell, Jeannelyn S. Estrella, Roy-Chowdhuri Sinchita, Fatemeh G. Amlashi, Zhenning Wang, Shumei Song, Jaffer A. Ajani. Overexpression of SHH and GLI1 contributes to poor prognosis and peritoneal metastases in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1071.