LAUSR

Objective: Uterine serous cancer (USC) represents about 10% of all uterine cancers, yet it accounts for up to 40% of all uterine cancer deaths. This lethality is due in part to the highly aggressive clinical course of this disease. Recently, galectin 3 (Gal3), a glycoprotein, was shown to contribute to malignant features in other tumors including ovarian cancer. In this study, we test the hypothesis that Gal3 facilitates malignant features in USC and might provide an opportunity for therapeutic intervention. Methods: The TCGA database was used to define the relationship between LGALS3 mRNA expression and prognosis. Gal3 knockout cells were established via CRISPR/Cas9-mediated deletion from USC cell lines, ARK1 and ARK2 (Gal3-KO). Gal3’s role in cell proliferation, cell cycle, and cell death were assessed. Gal3’s influence on apoptotic priming was done by BH3 profiling. Gal3 small molecule inhibitors (SMIs) were used to as an orthogonal approach to genetic knockdown. The effect of Gal3 loss on migration, invasion, and angiogenesis was assessed with transwell, Matrigel-invasion and HUVEC (human umbilical vein endothelial cells) tube-forming assays. The impact of Gal3 loss on cancer stem cells (CSC), phenotypically characterized by CD44, CD117, and ALDH activity, was measured by flow cytometry, and functionally by colony-forming and sphere-forming assays. An in vivo limiting dilution tumorigenic assay was performed with ARK1 Gal3-CTRL and KO cells in immunocompromised mice. Statistical analysis was done and a significance level of p < 0.05 was used. Results: The TCGA database revealed a worse prognosis for patients with USC and high LGALS3 mRNA. Cell Counts revealed Gal3-KO cells proliferated at a slower rate compared to Gal3-CTRL cells, and the difference wasn’t attributed to an increase in cell death. Cell cycle analysis showed an extended G2/M arrest in Gal3-KO cells suggesting Gal3 may influence cell cycle checkpoints. Unlike SKOV3 ovarian cancer cells, loss of Gal3 did not affect apoptotic sensitivity of USC cells as determined by BH3 profiling. Gal3 knockout reduced the number of migrating and invading cells in vitro. Gal3 SMIs (GB1107 and TD139) often differed in their response when compared to the knockout strategy. Conditioned media (CM) from ARK2 Gal3-CTRL promoted tube formation and invasion of HUVECs. This effect was reduced with CM from ARK2 Gal3-KO cells, suggesting Gal3 influences the tumor vascular microenvironment. Gal3 loss markedly reduced CD117+ cells and cells displaying high ALDH activity. Functionally, these findings were supported by a reduction in the number and size of colonies and fewer spheres formed by Gal3-KO cells compared to Gal3-CTRL cells. ARK1 Gal3-KO cells formed fewer and smaller tumors compared to Gal3-CTRL in mice. Conclusion: Gal3 has the potential to promote the more aggressive features of USC and patients would likely benefit from an anti-Gal3 based strategy. Citation Format: Yusuke Matoba, Dominique T. Zarrella, Venkatesh Pooladanda, Eugene Kim, Shaan Kumar, Mengyao Xu, Xingping Qin, Raj Kumar, Artem Kononenko, Irva Veillard, Kristopher Sarosiek, Oladapo Yeku, David R. Spriggs, Bo R. Rueda. Loss of galectin 3 reveals its potential roles in the aggressive pathology of uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2438.