Abstract B27: Identification of uveal melanoma disseminated cancer cell dormancy mechanisms
Authors: Lopez-Anton Melisa , Farias Eduardo F. , Chua Vivian , Terai Mizue , Nobre Anna Rita , Wedegaertner Philip , Sato Takami , Benovic Jeffrey , Aplin Andrew , Aguirre-Ghiso Julio 
Published: September 2020

Abstract
Introduction: Uveal melanoma (UM) is the most common intraocular cancer. Most patients succumb to liver metastasis after long asymptomatic periods through unknown mechanisms. After primary tumor resection, UM disseminated cancer cells (DCCs) remain dormant more than 15 years before growing lethal liver metastases. We propose that UM DCC dormancy may be responsible for the time delay to liver metastasis observed in clinic, which may represent an opportunity to prevent UM recurrences.
Methods: To characterize dormant and proliferative UM disseminated cells in the liver, we used an in vivo mouse model in which UM cells injected into the spleen disseminate and colonize the liver. In this model, UM DCCs stay dormant (up to 3 months) until some DCCs form proliferative metastasis. Liver DCCs that stayed dormant or formed metastasis were cell sorted and studied via gene transcription RNAseq. In addition, we monitored NR2F1, an orphan nuclear receptor of retinoic acid (RA) family, RA receptors (RARs) and RAR antagonist PRAME along with the YAP-TAZ pathway, which are linked to UM progression.
Results: Our data indicate that RA signaling may play a critical role in UM DCCs switch to become dormant. RARs α, β, and γ mediate transcriptional regulation upon RA ligand binding and are critical for the correct balance between self-renewal and differentiation of tissue stem cells. RA is highly abundant in the liver tumor microenvironment (TME). We found that RA regulated genes such as RARβ and NR2F1 are upregulated in UM single cells (UMsc), which are low for proliferation markers (Ki67) compared to large metastases (UMm). Our data indicate that NR2F1 is growth suppressor in UM organoid cultures, and that RARβ and NR2F1 are key mediators in UM DCCs dormancy. We also observed a downregulation of PRAME, a poor-prognosis indicator in UM patients. UMsc were RARβhigh, NR2F1high, and PRAMElow; conversely, UMm were RARβlow, NR2F1low and PRAMEhigh. We propose that upon liver colonization those DCCs able to sense RA in the TME activate RA signaling and switch off growth while engaging dormancy (RARβhigh, NR2F1high, PRAMElow) pathways. In vitro knockdown of NR2F1 in UM cell line with Gαq/11 mutation leads to a decrease in dormancy markers and an increase in YAP downstream targets, indicating an interplay between NR2F1 and the Hippo signaling pathway. Importantly, the use of retinoids and 5-azacytidine or the use of Gαq/11 inhibitors on UM cells in organoid cultures was able to turn on a dormancy program associated with melanocytic or neuritic differentiation.
Conclusion: Our data allow to conclude that UM DCCs dormancy may be linked to the activation of dormancy pathways including enhanced NR2F1 and RARβ signaling and the downregulation of pathways linked to poor prognosis (PRAMEhigh) and downstream signaling of Gαq/11 through YAP-TAZ transcriptional programs.
Citation Format: Melisa Lopez-Anton, Eduardo F. Farias, Vivian Chua, Mizue Terai, Anna Rita Nobre, Philip Wedegaertner, Takami Sato, Jeffrey Benovic, Andrew Aplin, Julio Aguirre-Ghiso. Identification of uveal melanoma disseminated cancer cell dormancy mechanisms [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B27.