Abstract 2942: ROR1: A novel target in endometrial cancer

Abstract

Background Endometrial cancer is one of the fastest rising cancers in women, and despite overall high survival, new therapies are required for women with aggressive subtypes of the disease. ROR1 and ROR2 are Wnt receptor tyrosine kinases whose expression is altered in a range of cancers. This study aimed to investigate the role of ROR1/2 in endometrial cancer (EC). Method Immunohistochemistry (IHC) of ROR1 and ROR2 was performed on an EC patient cohort (Australia-wide population-based Australian EC Study, ANECS, n=499). The association between ROR1/2 staining intensity (0-3) and clinicopathological parameters was analyzed, as well as association with 5-year progression free survival (PFS) and overall survival (OS). Cox multivariate regression was also applied to analyze the impact of selected covariates (age, BMI, FIGO stage, grade and subtypes) on the PFS and OS. The functional effect of ROR1 and ROR2 modulation was further investigated in two EC cell lines–KLE (high ROR1, low ROR2) and MFE-296 (low ROR1, high ROR2). For KLE, ROR1 silencing and ROR2 overexpression were performed. In contrast, ROR2 silencing and ROR1 overexpression were performed in MFE-296. ROR1 or ROR2 silencing was achieved via transfection with ROR1 or ROR2 siRNA. ROR1 pCMV3 plasmid or ROR2 pFLAG plasmid were transfected for ROR1 or ROR2 overexpression. All the conditions were compared to the negative control which was prepared by transfecting both non-targeting siRNA and pCMV3-NH plasmid. Effects on proliferation, adhesion, migration and invasion were measured. Result In the patient cohort, ROR1 expression level was significantly associated with tumor grade (p=0.004) and moderately associated with FIGO stage (p=0.057). A significant decrease in OS and PFS was observed in patients with high ROR1 expression (p=0.045 and 0.003 respectively). This correlation was not lost when filtering against multiple parameters including age, BMI, FIGO stage and tumor grade in COX regression (p=0.049). No significant correlation was observed for ROR2 expression with OS or PFS, though high ROR2 showed a trend towards better PFS. ROR1 downregulation or ROR2 upregulation in KLE cells decreased cell proliferation after 72 hours. The combination of the two further reduced cell proliferation significantly after 48h and 72h (p=0.04 and 0.004 respectively). Proliferation was not significantly altered in MFE-296, although ROR1 overexpression showed a trend for increasing cell proliferation after 72h. ROR1 silencing in KLE decreased the migration ability moderately (p=0.059) and its combination with ROR2 overexpression further reduced migration significantly (p=0.038). No significant change in invasion or adhesion was observed. Conclusion This study confirms the oncogenic role ROR1 plays in EC, which warrants the future application of ROR1-targeting therapies in EC patients. The role of ROR2 is more complex in EC and may be context dependent. Citation Format: Dongli Liu, Kate Gunther, Benjamin Daniels, Tracy O'Mara, Katrina Tang, Australian National Endometrial Cancer Study Group, Caroline Ford. ROR1: A novel target in endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2942.