Evans, Bradley J.

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

Abstract Background: Most ovarian cancer (OC) patients present with advanced disease and ultimately develop chemo-resistant relapses. Stroma plays a significant role in OC behavior including invasiveness, fibrosis and chemoresistance. TGF-β is important for crosstalk between stroma and cancer cells, and stromal activation in cancer has similarities to matrix remodeling in fibrosis. We have shown that TGF-β and fibrosis-inhibitors can suppress OC cell proliferation, migration and invasion. Such inhibitors are available for clinical use, and we have begun to test their efficacy in OC in vivo. Methods: Animal experiments were approved by Mayo Clinic IACUC. Intraperitoneal xenograft models were derived from OVCAR8 (5×106) cells (OV8-CDX) or 0.1 gm OC patient tumor tissue (PDX) in NSG mice. OV8-CDX and PDX PH003 (an aggressive, chemoresistant and ascites-prone model) (n=5-10 mice/arm) were treated with 2 agents and compared to untreated controls (C): 1) C vs. the fibrosis inhibitor Pirfenidone (P, 200 mg/kg, BID, provided by Genentech); 2) C vs. TGF-β receptor I inhibitor Galunisertib (G, 75 mg/kg, BID, provided by Eli Lily). Mice were pretreated for 2 days before xenografting and treated for 4 weeks with ultrasound (US) to measure tumor growth. At day (d) 28, we i) collected tumor (OV8-CDX); or ii) observed until progression (PH003, PH053). OV8-CDX tumors were used for target gene analysis. Results: P treatment delayed tumor growth in OV8-CDX relative to control (C): tumors established in 0/5 (P) vs. 1/5 (C), and 1/5 (P) vs. 4/5 (C) of mice at d14 and d28, respectively. The reduced tumor weight (g) at d28 was also confirmed (0.20±0.10 vs. 0.54±0.23, p=0.02). Similarly, P treatment in PH003 inhibited the tumor growth (tumor area (cm2) at d28: 0.74±0.43 vs. 1.04±0.51, p=0.28), ascites development (volume (ml): 1.14±0.54 vs. 3.37±0.62, p=0.05) and prolonged survival time (p=0.04). G treatment was also associated with delayed tumor establishment in OV8-CDX relative to C: tumors presented in 0/5 (G) vs. 1/5 (C), and 1/5 (G) vs. 4/5 (C) of mice at d14 and d28, respectively. Associated d28 tumor weight was significantly reduced (0.20±0.10 vs. 0.54±0.23, p=0.02) as well. For PH003, G treatment was associated with delayed tumor growth (tumor area at d28: 0.57±0.27 vs. 1.04±0.51, p=0.05), a marked reduction in ascites (volume: 1.25±0.56 vs. 3.37±0.62, p=0.05) and a prolonged survival time (p=0.04) We identified down-regulation of TGF-β signaling-related genes (e.g. TGFB1, TGFBR2) and fibrosis-related biomarkers (e.g. FAP, COL11A1) following drug treatment confirming inhibition of relevant pathways. Conclusion: We report that 2 currently available inhibitors targeting either fibrosis or TGF-β appear to have efficacy in vivo and delay tumor growth in OC PDX models. These results suggest promise for a strategy of targeting the stromal component of OC. Citation Format: Qing Zhang, Xiaonan Hou, Bradley J. Evans, John J. Weroha, William A. Cliby. Inhibitors of fibrosis and TGF-beta delay tumor xenograft growth and reduce ascites in ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2017-961