Background Uterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3’s role in promoting USC pathology is lacking. Methods We explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3’s impact on cell function. Results TCGA analysis revealed a worse prognosis for USC patients with high LGALS3 . Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids. Conclusion These findings suggest inhibiting Gal3 may benefit USC patients.

Abstract Ovarian cancer stem cells (CSCs) are thought to serve as seed cells for recurrent drug resistant disease. Extracellular vesicles (EVs) derived from CSCs are known to promote a more favorable tumor microenvironment. However, there is limited research focused on the impact of EV exchange between tumor cells. We hypothesized that CSC enriched tumor cell fractions could confer their stem like properties to chemo-sensitive non-CSCs via EVs. To test our hypothesis, we utilized established ovarian cancer (OvCa) cell lines (SKOV3, A2780, and UWB1.289 olaparib sensitive (U-OlaSen) and resistant (U-OlaRes)) and high grade serous ovarian cancer (HGSOC) patient derived organoid (PDO) lines. Small EVs were isolated via ultra-filtration and centrifugation, validated, and quantified. EVs from CSC enriched cultures, PARP inhibitor resistant lines, or drug treated (olaparib or carboplatin) lines were cultured with treatment naïve or treatment sensitive lines for defined time points. The endpoints included assessment of changes in ALDH activity (ALDEFLUOR™ Kit) and cell viability (Annexin V staining) using flow cytometry. In addition, relative levels of Enhancer of Zeste Homologue 2 (EZH2), Histone 3 lysine 27 trimethylation (H3k27me3), Disrupted Meiotic cDNA recombinase 1 (DMC1), phosphorylated and non-phosphorylated CHK1 were determined by western blotting. Drug resistance was determined post exposure of the more sensitive cells to EVs derived from the more resistant cells by treatment by either carboplatin or olaparib. At least 3 independent experiments were completed for each endpoint. Statistical analysis was performed by GraphPad Prism and p < 0.05 was considered as statistically significant. Exposure of small EVs (2.5 ug/ml) derived from CSC enriched cultures or drug resistant cell fractions to the more sensitive tumor cell populations resulted in a marked increase in ALDH activity, indicating an increase in CSC markers. This increase was concurrent with an EV induced increase in the relative levels of EZH2, DMC1, total and phosphorylated CHK1 compared to controls. EVs derived from tumor cells treated with carboplatin or olaparib promoted resistance in the more sensitive tumor cell cultures further implicating the ability to exchange resistance properties. Similar results were observed in response to EVs from known drug resistant human HGSOC organoids on U-OlaSen cells. In summary, these findings provide evidence that EVs from CSC enriched fractions can confer CSC like properties via canonical and non-canonical signaling to the more sensitive tumor cells. We conclude that in addition to the concept that tumor cells can promote drug resistance by diluting cytotoxics among neighboring tumor cells, the more resistant CSCs can promote stem like activity in neighboring drug sensitive tumor cells allowing them to overcome anticancer drug treatment and ensuring their survival and adaptation. Citation Format: Venkatesh Pooladanda, Shaan Kumar, Yusuke Matoba, Maryam Azimi Mohammadabadi, Dominique T. Zarrella, Ursula A. Winter, Sarah J. Hill, Hyungsoon Im, Bo R. Rueda. Extracellular vesicles derived from enriched ovarian cancer stem cell fractions promote stem like properties in the more vulnerable tumor cell populations [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A113.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2 . Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

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.