Endometrial Cancer Cell Lines Research Articles

Exploring etofenamate hydrazide-hydrazone/copper(II) complexes: Synthesis, anticancer activity, carbonic anhydrase IX inhibition and docking studies

Hydrazide-hydrazone derivatives have garnered significant interest from researchers globally due to their wide range of biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, a novel series of etofenamate hydrazide-hydrazone compounds (2a-2s) and their Cu(II) complexes (3a-3s) were designed and synthesized. The compounds were characterized using various analytical techniques such as FT-IR, 1H NMR, 13C NMR, MS, and elemental analysis. Additionally, the compound 2c and Cu(II) hydrazone complex 3a were further characterized using single X-ray crystallography. The anti-proliferative activity of the compounds was evaluated against Ishikawa human endometrial cancer cell line and non-tumour L929 cells using MTT assay. Additionally, the apoptotic potential of the compounds was investigated through caspase-3 activity, Bax and Bcl-2 gene expression analysis, and annexin-V binding. Furthermore, carbonic anhydrase IX activity and in silico studies were conducted to elucidate the mechanism of action. Overall, compound 3s demonstrated significant antiproliferative effects with an IC50 value of 0.27±0.01 µM against Ishikawa cells.

17β-estradiol Inhibits Oxidative Stress-Induced Apoptosis in Endometrial Cancer Cells by Promoting FOXM1 Expression

17β-estradiol Inhibits Oxidative Stress-Induced Apoptosis in Endometrial Cancer Cells by Promoting FOXM1 Expression

Upregulation of ESPL1 is associated with poor prognostic outcomes in endometrial cancer

Background Extra spindle pole bodies-like 1 (ESPL1) is known to play a crucial role in the segregation of sister chromatids during mitosis. Overexpression of ESPL1 is considered to have oncogenic effects in various human cancers. However, the specific biological function of ESPL1 in endometrial cancer (EC) remains unclear. Methods The TCGA and GEO databases were utilized to assess the expression of ESPL1 in EC. Immunohistochemistry was utilized to detect separase expression in EC samples. Kaplan-Meier survival analysis and Cox regression analysis were performed to evaluate the diagnostic and prognostic significance of ESPL1 in EC. Gene Set Enrichment Analysis (GSEA) was employed to explore the potential signaling pathway of ESPL1 in EC. Cell proliferation and colony formation ability were analyzed using CCK-8 and colony formation assay. Results Our analysis revealed that ESPL1 is significantly upregulated in EC, and its overexpression is associated with advanced clinical characteristics and unfavourable prognostic outcomes. Suppression of ESPL1 attenuated proliferation of EC cell line. Conclusion The upregulation of ESPL1 is associated with advanced disease and poor prognosis in EC patients. These findings suggest that ESPL1 has the potential to serve as a diagnostic and prognostic biomarker in EC, highlighting its significance in the management of EC patients.

Abstract 7563: Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line

Abstract Objectives: PARP inhibitor is effective in treating DNA repair deficiency endometrial cancer. Endometrial cancer (EC) is one of the most common gynecological cancers in developed countries and its incidence is increasing, and the prognosis of patients with early stage and low-risk EC is very good, but the prognosis of progressive and recurrent EC is poor. Novel PARP inhibitor JPI-547 is a PARP and tankyrase dual inhibitor, and in cancer cells, tankyrase inhibitor plays an important role in DNA repair, telomere maintenance, and cell survival. This experiment was conducted to determine whether the reactivity of the PTEN mutant EEC was increased by using the Novel PARP inhibitor. Method: Human endometrioid endometrial cancer cell lines, Hec-1A, Hec-1B and Ishikawa were obtained. Cell viability assay for the drugs Olaparib and JPI-547 were assessed. Colonies consisting of at least 50 cells were counted. The number of dead cells was counted in five different areas of the hemocytometer, and the experiment was performed three times. The percentage of dead cells was analyzed compared to the untreated group. We confirmed the efficacy by siRNA transfection and immunofluorescence. Result: When treated with Olaparib, there was no change in cell viability in the rest of the cells except for Ishikawa cells, which are PTEN mutations. It decreased by 35.4% in Ishikawa cells, whereas when treated with JPI-547, all cell lines Hec-1A, Hec-1B, and Ishikawa cell viability decreased by 28%, 34.3%, and 48.7%, respectively. PTEN knock-down reduced cell visibility by 28% when treated with olaparib, whereas when treated with JPI-547, Hec-1A (siCont) and Hec-1A (siPTEN) decreased by 26% and 42%, respectively, in all cells regardless of whether PTEN knock-down is present. When treated with Olaparib, dead cells were identified only in PTEN knock-down cells, increasing by 22%. When treated with JPI-547, dead cells increased by 25.5% and 33% in all cells of Hec-1A (siCont) and Hec-1A (siPTEN), respectively. It was confirmed that JPI-547 can reduce cell viability regardless of the formation of Rad51 according to PTEN. Conclusion: JPI-547, a novel PARP inhibitor, showed good efficacy in PTEN mutation and PTEN-wild-type EEC cell lines than in conventional PARP inhibitor. The effect of reducing survival of ECC cell line with JPI-547 was confirmed with or without PTEN mutation. Further evaluation and in vivo experiments are needed to prove the efficacy of JPI-547 in EC. Citation Format: Shin-Wha Lee, So-Hyun Nam, Su-Bin Park. Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7563.

Abstract 249: Deciphering the impact of TRIB3-ELF4 interaction in regulating CDK6 on cancer stem cell activity in endometrial cancer

Abstract Background: Endometrial cancer (EC) is the most common gynecological tumor among women worldwide, posing a serious threat to women's health. ELF4 (E74 like ETS transcription factor 4), a member of the ETS family, plays a role in facilitating cancer progression. Our previous work demonstrated the role of direct binding between ELF4 and tribbles pseudokinase 3 (TRIB3) in promoting β-catenin expression. In this study, we further explore ELF4's involvement in the maintenance of cancer stem cells (CSCs) in EC. Methods: The vital role of ELF4 in EC was determined through gain- and loss-of-function assays. ELF4 was silenced in two EC cell lines, AN3CA and HEC-1A, and primary EC cells from a Taiwanese patient (EMC6), using RNA interference techniques. The expression of ELF4, TRIB3, and CDK6 in human EC samples was assessed using immunohistochemistry. Tumorsphere formation assays, quantitative real-time PCR, and immunoblotting quantified CSC activity and the involvement of related signaling pathways. Transcriptional regulation associated with ELF4 was investigated using luciferase reporter assays and chromatin immunoprecipitation. Results: Analysis of the TCGA database via the TISIDB web tool revealed that elevated ELF4 levels correlated with higher histological grades, advanced clinical stages, and poorer overall survival in EC patients. A grade-dependent increase in ELF4 expression was observed using tissue microarrays. Knockdown of ELF4 in EC cell lines and patient-derived EC cells significantly reduced cell proliferation, cell cycle progression, migration, invasion, and CSC activity. RNA-seq data analysis of ELF4-knockdown EC cells identified CDK6 as a potential downstream target of the ELF4/TRIB3 interaction. Knockdown of ELF4 and TRIB3 significantly downregulated CDK6 expression. Co-expression of TRIB3 and ELF4 enhanced CDK6 expression, and their expression was positively associated with CDK6 in EC specimens. We also demonstrated that CDK6 facilitates EC-CSC activity. Knockdown of CDK6 significantly attenuated tumorsphere formation and reduced the levels of cancer stemness-related proteins, including OCT4, NANOG, and c-MYC. Conclusion: Our study demonstrates the essential roles of ELF4 and TRIB3 in regulating CDK6 expression, contributing to the maintenance of EC-CSCs. Citation Format: Wen-Ling Wang, Wen-Wei Chang. Deciphering the impact of TRIB3-ELF4 interaction in regulating CDK6 on cancer stem cell activity in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 249.

Abstract 5089: Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates

Abstract Background: Endometrial cancer is the most frequently occurring gynecologic cancer in developed countries and the third leading cause of cancer deaths among women. It remains one of the few malignant tumors with rapidly increasing mortality and morbidity. Patients with recurrent or metastatic endometrial cancer continue to experience poor outcomes. Antibody-drug conjugates (ADCs) represent a class of targeted therapeutic modalities that offer biomarker-based therapeutic options for patients with endometrial cancer. In this study, we proposed to identify a novel molecular target for the preclinical development of an ADC for endometrial cancer-targeted therapy. Methods: Firstly, to determine the level of target expression within endometrial cancer tissue components as well as differences, we performed immunohistochemical staining of human endometrial cancer and paracancerous tissue. Secondly, the expression of the target on the cell membrane surface was quantitated and qualitatively verified using flow cytometry and single-photon confocal microscopy. In the third step, an endocytosis assay was used to verify the endocytosis of the target antibody by EC cells. Confocal imaging can directly observe the endocytosis of the target antibody, and flow cytometry can be analyzed and quantified. Co-localization experiments with antibody fluorescence can verify the specific site where the target antibody is endocytosed into cancer cells. We then designed and characterized two targeted ADCs. Next, the in vitro inhibitory activities of these ADCs against endometrial cancer cell lines and normal cells were evaluated using the CCK8 assay. Finally, we established a cell line-derived xenograft (CDX) model of endometrial cancer to validate the in vivo efficacy and bio-safety of the ADCs. Results: We have determined a correlation between ICAM1 overexpression and EC, and IHC staining of patient tissues showed significantly higher levels of ICAM1 expression in endometrial cancer tissues than in paracancerous tissues. The high cell surface expression of ICAM1 was verified in a panel of various human endometrial cancer cell lines. Moreover, two ICAM1 ADCs were established using clinically-approved ADC linker and payload combinations. It was visually confirmed that ICAM1 ADCs selectively recognize endometrial tumors and efficiently enter endometrial cells via antigen-mediated endocytosis. We compared the anti-tumor activity of ICAM1 ADCs with standard-of-care chemodrugs, and ICAM1 ADCs outperformed chemodrugs in the CDX model of endometrial cancer without toxicity. Conclusion: We identified that ICAM1 is a promising ADC target for endometrial cancer and confirmed the potent anti-tumor activity and biosafety of ICAM1 ADCs in vitro and in vivo, warranting further pre-clinical and clinical investigations. Citation Format: Hanfei Xie, Lu Sun, ShiLi Yao, Peng Guo, YingLi Zhang. Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5089.

Abstract 7061: Targeting metabolic vulnerabilities in ARID1A/B dual-deficient dedifferentiated endometrial carcinoma

Abstract Background: Dedifferentiated endometrial carcinoma (DDEC) is an aggressive endometrial carcinoma defined histologically by undifferentiated carcinoma juxtaposed against stage 1 or 2 endometrial adenocarcinoma. DDEC responds poorly to conventional platinum/taxane-based chemotherapy, particularly in the case of extrauterine spread. This highlights a need to develop novel therapies for individuals with DDEC. Genetically, a third of DDEC cases have co-inactivating mutations of ARID1A and ARID1B. These genes encode core subunits of the sucrose/switch non-fermentable (SWI/SNF) complex that regulate transcription. Furthermore, dual loss of ARID1A/B is believed to play a role in driving DDEC tumor development. We hypothesize that ARID1A/B dual-deficiency in DDEC generates unique genetic deficiencies that allow for the development of novel therapies for this cancer. We approached this hypothesis by first conducting a preliminary analysis of the Cancer Dependency Map (DepMap), which has identified vulnerabilities in mitochondrial functioning in ARID1A/B dual-deficient DDECs. As such, we aimed to understand the role of mitochondrial oxidative phosphorylation (OXPHOS) and redox homeostasis in ARID1A/B dual-deficient DDEC to create new treatment options. Methods: A panel of ARID1A/B dual-deficient and proficient endometrial cancer cell lines were treated with OXPHOS inhibitor IACS-010759 and reactive oxidative species (ROS)-inducing agent elesclomol to identify shifts in drug response between the two groups. To account for genomic differences between the cell lines, we also treated an ARID1B knock-out isogeneic cell line with the drugs. Additionally, changes in metabolism in the endometrial cancer cell lines were measured using Seahorse metabolic flux assays to determine if ARID1A/B dual-deficient cell lines have an increased dependency on OXPHOS for cell growth. Results: While the Seahorse assays revealed no significant difference in OXPHOS and glycolysis activities between the two groups, drug sensitivity assays demonstrated that ARID1A/B dual-deficient cancers are more sensitive to OXPHOS inhibition and increased ROS production than the proficient cell lines. Depletion of ARID1B in an ARID1A deficient cell line sensitizes the cells to both drugs. Furthermore, IACS-010759 significantly suppressed the growth of ARID1A/B-deficient DDEC xenograft tumor growth. Conclusion: ARID1A/B-dual deficient cancer cells rely on mitochondria function for survival. The underlying mechanisms are currently under investigation. Citation Format: Rebecca Ho, Eunice Li, Chae Young Shin, Shary Chen, David Huntsman, Yemin Wang. Targeting metabolic vulnerabilities in ARID1A/B dual-deficient dedifferentiated endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7061.

Abstract 1959: BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models

Abstract Background: Many cyclin-dependent kinases (CDK) bind regulatory cyclins to control cell cycle progression. Aberrant CDK2 activation occurs in selected cancers (e.g., ovarian cancer, endometrial cancer) through CCNE1 amplification, leading to Rb phosphorylation, inactivation, and cell cycle checkpoint dysregulation. CCNE1 amplification and subsequent cyclin E overexpression are associated with chemotherapy resistance and shorter progression-free and overall survival. Therefore, novel therapies are needed to inhibit aberrant CDK2 activation and elicit positive clinical benefit in these patients. BLU-222 is a potent, highly selective, orally bioavailable, investigational CDK2 inhibitor with demonstrated activity in preclinical CCNE1-amplified ovarian cancer models. Here, we explored additional, specific biomarkers to predict BLU-222 sensitivity in ovarian and endometrial cancer as monotherapy or in novel combination treatment strategies. Methods: In vitro antiproliferative effect of BLU-222 was assessed by CyQuant in a panel of CCNE1-aberrant or CCNE1-normal ovarian and endometrial cell lines. Combination of BLU-222 and paclitaxel was evaluated by dosing matrix; synergy was calculated using SynergyFinder. In vivo antitumor activity of BLU-222 as a single agent or combined with paclitaxel was measured in CCNE1-aberrant endometrial cancer patient-derived xenograft (PDX) models (XenoSTART). Results: In ovarian and endometrial cell lines, CCNE1 amplification (copy number ≥ 6) predicted strong antiproliferative response to BLU-222. Using multivariate biomarker analysis, high p16 protein expression and wild-type Rb protein were identified as additional predictive markers for response to BLU-222 in CCNE1-aberrant cell lines. BLU-222 with paclitaxel was broadly active in endometrial cancer cell lines, with the strongest combination effect in cells with high CCNE1 expression, wild-type Rb protein, and low p16 protein expression. In vivo, BLU-222 elicited strong monotherapy antitumor response in CCNE1 copy number-aberrant endometrial cancer PDX models, and this effect was strengthened with the combination of paclitaxel. Conclusions: In preclinical models of CCNE1-aberrant endometrial cancer, BLU-222 demonstrated enhanced activity in combination with paclitaxel. Furthermore, Rb and p16 protein expression can be used in conjunction with CCNE1 copy number to predict response to both BLU-222 monotherapy and combination treatment. These data provide a rationale for using BLU-222 in combination with paclitaxel and demonstrate the potential benefit of using specific biomarkers to predict response. Citation Format: Nealia House, Victoria Brown, Liang Yuan, Maxine Chen, Stephanie Lee, Rentian Wu, Lakshmi Muthuswamy, Scott Ribich, Philip Ramsden, Kerrie Faia. BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1959.

Abstract 3224: Identifying the molecular mechanisms underlying progesterone receptor downregulation in endometrial cancer

Abstract Background: Endometrial cancer (EC) is the most common gynecologic cancer. Cancer survival has improved over the past four decades for all the most common cancers except cervical and EC, calling for significant treatment advancements. Progesterone receptor (PR) is a favorable prognostic marker for EC and other solid tumors. However, PR expression has been shown to be reduced or lost in these malignant tumors, which correlates with worse patient survival and limits the effectiveness of progestin therapy. Thus, restoring functional PR expression is vital to sensitizing tumor cells to progestin therapy, enhancing EC clinical interventions, and improving patient outcomes. In this project, we aim to restore functional PR expression. Methods: To visualize PR expression in real-time, we constructed an innovative endogenous PR reporter gene transfected EC cells. This system facilitated screening potential small molecular PR inducers from the FDA-approved oncology drug library. Sixteen drugs were identified to increase PR expression; topoisomerase II inhibitors (Top IIi), mitoxantrone and idarubicin were the top hits. Drug effect of Top IIi, as well as HDAC inhibitor romidepsin, dual PI3k/HDAC inhibitor CUDC-907, and PHB2 inhibitors, fluorizoline (FLZ) and rocaglamide (RocA), were evaluated as single drug or in combination. Potential PR repressors, including G9a, HDAC2, PHB2, and Top2A, were knocked down using siRNA, shRNA, or CRISPR-Cas9 in EC cells and PR expression was analyzed. A chromatin immunoprecipitation (ChIP) assay was also conducted to validate bona fide PR repressors. Results: Mitoxantrone and idarubicin were validated to be effective PR inducers. G9a, HDAC2, PHB2, and Top2A were confirmed PR repressors. Single treatment with mitoxantrone, idarubicin, romidepsin, CUDC-907, FLZ, and RocA achieved effective inhibition of EC cell proliferation and enhanced PR expression except FLZ and RocA. Combined romidepsin with Topi or PHB2i demonstrated additive or synergistic effects. More importantly, dual treatment of CUDC-907 with mitoxantrone enhanced PR expression compared to a single treatment or no treatment control. Our mass spectrometry and ChIP assay by specific antibodies against HDAC2 and PHB2 revealed that potential mechanisms include PHB2 forming a complex with HDAC2 that binds on the PR promoter, thus inhibiting PR transcription. Conclusion: Co-treatment of CUDC-907 with Top2Ai increased PR expression and enhanced cell death in EC cell lines, indicating a potential treatment strategy. In vivo studies utilizing our EC-PDX models will further validate the efficacy of restoring PR expression and suppressing EC tumor growth. Further studies of the molecular mechanisms behind this strategy will be conducted. Citation Format: Xiangbing Meng, Lillie Lamont, Shujie Yang. Identifying the molecular mechanisms underlying progesterone receptor downregulation in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3224.

Abstract 98: SETDB1 promotes tumorigenesis in endometrial cancer through suppressing anti-tumor immune response and increasing proliferation

Abstract Background: An H3K9 methyltransferase and cancer driver gene, SETDB1, promotes tumorigenesis in many cancer types, such as melanoma, and breast cancer. SETDB1 is often overexpressed and correlates with worse survival. Multiple mechanisms have been reported for its oncogenesis from promoting tumorigenesis and regulating immune evasion. The roles of SETDB1 in driving endometrial cancer (EC) progression is not well-studied. Methods: EC cell lines were used for knockout SETDB1 using CRISPR-Cas9 system, RNA-seq was conducted to discover SETDB1 regulated genes. ChIP-qPCR was performed to identify SETDB1 direct targets. Immunocompromised NSG and immunocompetent C57BL/6 mice were utilized to investigate SETDB1 promoted tumor growth, immune evasion, and mice survival. IHC and flow cytometry was conducted to evaluate macrophage infiltration. Cytokines were screened by qPCR for potential macrophage chemoattractant. Results: In vitro, knockout SETDB1 decreased cell proliferation and enhanced G1/S cell cycle arrest. RNA-seq data revealed that in SETDB1−/− clones, many important oncogenes such as POLR2A, MUC5AC, and MKI67 were downregulated, while tumor suppressors such as PGR, RERG, and ZNF582 were upregulated. ChIP assay discovered that SETDB1 directly binds and represses ZNF582, ZNF266, ZNF841 expression via depositing H3K9me3 at promoter region. In vivo, SETDB1−/- significantly reduced tumor growth and prolonged NSG mice survival up to 100 days. SETDB1 promoted tumor growth via 1. Intrinsic: increasing mitotic figure, pH3-S10 and Ki67 level, 2. Extrinsic: blocking macrophage infiltration and chemokine secretion. Our in vitro functionality assay confirmed that bone-marrow derived M1-polarized macrophages promoted greater killing for SETDB1−/− cells. We surveyed 17 cytokines and found CCL5, CXCL9, and IL6 mRNA expression were enhanced in SETDB1−/− tumors suggesting active immune response. Consistent with the elevated CCL5 mRNA expression, CCL5 protein level was also upregulated in macrophages and cancer cells in SETDB1−/- tumors. To prove SETDB1 directly regulates CCL5 expression, we re-expressed SETDB1 in SETDB1−/− cells, and indeed, the SETDB1 re-expression did suppress CCL5 expression. Using immunocompetent C57BL/6 mice, we further confirmed that SETDB1 promotes tumor growth of MSH2-369 mice cells and blocks macrophage infiltration. To understand SETDB1 regulated pathways in EC patients’ samples, using EC-TCGA data, we discovered that high SETDB1 expression correlated with higher level of active transcription genes and reduced immune response genes. Our findings provide the mechanisms behind these observations. Conclusion: SETDB1 drives EC tumorigenesis by both intrinsically upregulating cell proliferation genes and promoting immune escape extrinsically. Our novel findings suggest SETDB1 is a promising anticancer target for EC patients. Citation Format: Kiarash Salari, Jiaqing Hao, Matthew Wells, Eleanor Johnson, Ryan Jilek, Jack Gilbert, Ryan Mclerran, David Meyerholz, Melinda Yates, Bing Li, Shujie Yang. SETDB1 promotes tumorigenesis in endometrial cancer through suppressing anti-tumor immune response and increasing proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 98.

Abstract 3369: Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage

Abstract Background: Endometrial cancer (EC) is the most common gynecologic malignancy with increasing incidence and mortality in the U.S. Among the four subtypes characterized by the Cancer Genome Atlas, the serous-like copy number high (CNH) subtype which occurs in 15-25% of EC is associated with poor prognosis and platinum resistance, requiring alternative treatment options. ~85% CNH EC cases have impaired G1/S checkpoint regulation, making cells rely on ATR-mediated G2/M cell cycle checkpoints for optimal DNA replication and repair. Additionally, over 80% of ECs exhibit PI3K pathway activation (e.g., PIK3CA mutations), linked to enhanced DNA repair and drug resistance. Therefore, we hypothesized that dual ATR and PI3Kα inhibition would enhance DNA damage, increasing cell death in EC. Methods: CNH EC cell lines included platinum-resistant (KLE, MFE280, HEC1A, ARK2) as well as platinum-sensitive (ARK1) lines used to evaluate the activity of ATR inhibitor (ATRi) camonsertib and PI3Kα inhibitor (PI3Kαi) inavolisib. Cell growth was assessed by 3-day XTT and 9-12-day colony-forming assays. The degree of combination synergy, additivity, or antagonism was calculated using SynergyFinder with a reference highest single agent (HSA) model. An HSA synergy score > 10 indicates synergy, -10 to 10 suggests additivity and < -10 represents antagonism. DNA damage endpoints were measured by alkaline comet assay and immunoblotting for γ-H2AX. Data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P < 0.05 was statistically significant. Results: All tested EC cell lines showed sensitivity to clinically attainable doses of ATRi monotherapy (IC50 0.09 - 0.98 μM), but varying sensitivity to PI3Kαi monotherapy (IC50 0.03 - 67.01 μM) unrelated to their PIK3CA mutation status. Combination treatment using clinically attainable concentrations (camonsertib < 6 μM; inavolisib < 10 μM) yielded additivity in all cell lines independent of platinum sensitivity (HSA synergy scores -0.8-10.0). Notably, increased cytotoxic effects were observed in both PIK3CA wild-type (KLE) and PIK3CA-mutant (MFE280 and HEC1A) platinum-resistant lines by colony-forming assays (4.8- to 15.4-fold decrease in colony-forming ability relative to ATRi; 1.5- to 3.5-fold decrease relative to PI3Kαi). ATRi and PI3Kαi combination induced greater DNA damage, evidenced by elevated mean comet tail moment relative to ATRi (increased 2.5- to 6.7-fold; P < 0.01) or PI3Kαi (increased 2.1- to 5.6-fold; P < 0.01) regardless of PIK3CA mutation status. Increased γ-H2AX levels were found in HEC1A cells with combination treatment (3.2-fold increase relative to ATRi; 16-fold increase relative to PI3Kαi). Conclusion: Our results suggest that dual inhibition of ATR and PI3K pathways induces greater cell death by increasing DNA damage in platinum-resistant EC cells independent of PIK3CA mutation status. Citation Format: Kristen R. Ibanez, Tzu-Ting Huang, Sotirios Sotiriou, Yvonne G. Lin, Jung-Min Lee. Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3369.

Abstract 6707: IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors

Abstract Ovarian cancer is characterized by late stage disease and a poor prognosis. Endometrial cancer is a heterogeneous disease with different prognoses depending on the molecular subtype. Novel treatment strategies are needed to improve patient survival in both entities. T cell-based immunotherapy has significantly improved the treatment options for many malignancies. B7-H3 (CD276) is an interesting target for immunotherapeutic approaches because it is overexpressed on a wide range of tumor entities and has characteristics of immune checkpoint molecules. Its expression not only on tumor cells but also on the tumor vasculature allows improved infiltration of immune effector cells into the tumor to overcome the major limitation of T cell-based therapy of solid tumors, which is the access of immune cells to the tumor site. A novel bispecific antibody in an IgG-based format called CC-3 with B7-H3xCD3 specificity has already been shown to efficiently induce T cell responses against gastrointestinal malignancies. Here we show that B7-H3 is highly expressed on endometrial and ovarian cancer cell lines. Treatment with CC-3 induced T cell activation and degranulation as well as secretion of IL-2, IFNy and perforin. These results demonstrate CC-3 induced T cell reactivity against ovarian and endometrial cancer cells. In addition, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. This resulted in potent target cell lysis. Taken together, our results underscore the potential of CC-3, which is currently in GMP manufacturing, to enable clinical evaluation for the treatment of gynecologic malignancies. Citation Format: Sarah Maria Greiner, Jonas Mauermann, Martina Lutz, Ilona Hagelstein, Engler Tobias, André Koch, Hartkopf Andreas, Brucker Sara, Latifa Zekri, Melanie Märklin. IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6707.

Abstract 559: Investigating DDX3X as a target to improve outcomes in Black and Native American endometrial cancer patients

Abstract Introduction: The goal of this project was to identify and test a new molecular target that will help reduce the worse outcomes of endometrial cancer, especially in Black and Native American patients. Endometrial cancer is rapidly on the rise in the developed world. It has overtaken many other types of cancer in both incidence and mortality. Black populations are disproportionately impacted by endometrial cancer, both in Oklahoma and across the United States. In Oklahoma specifically, Native Americans are the most heavily impacted group. Methods: Machine learning techniques were used to analyze comprehensive metabolic panel (CMP) data from electronic medical record of patients in the University of Oklahoma (OU) network of hospitals and clinics. This data was broken down by endometrial cancer status, race, and age. Plasma samples of endometrial cancer patients were collected from the OU Health Sciences Center Biobank. These samples were analyzed by mass spectrometry. Endometrial cancer cell lines Ishikawa (low-grade) and ARK-1 and ARK-4 (high grade) were cultured in EMEM. For treating the cells, we used RK-33, a small molecule inhibitor of DDX3X. MTT assays were performed in 96-well plates at increasing concentrations of RK-33 for 48 hours. Western blots were used to determine effect of the drug on cellular DDX3X and yH2AX protein levels. Results: Machine learning analysis EMR data revealed a difference between races in albumin levels of women with endometrial cancer. Black and Native American patients had lower albumin, which is known to correlate to worse outcomes. IPA analysis of plasma proteomics revealed several proteins that were differentially expressed between races. Among these was DDX3X, the X-linked form of DDX3, a DEAD-box RNA helicase which is involved in DNA damage repair. Patient data indicated that patients with high DDX3X expression tended to have lower survival rates. MTT analysis determined that Ishikawa has a lower IC50 than the high-grade cell lines, but all responded. Western blotting confirmed that treatment with RK-33 reduced the amount of DDX3X in the cells and increased y-H2AX, indicating more DNA damage. Conclusions: This study demonstrates that DDX3X is a valid molecular target in endometrial cancer. The differential expression across race groups indicates that Black and Native Americans may stand to benefit the most from a novel drug targeting this difference. Because DDX3X is involved in DNA damage repair, combining the treatment with a DNA-damaging drug may increase response to the drug as well. Citation Format: James D. Lausen, Doris M. Benbrook. Investigating DDX3X as a target to improve outcomes in Black and Native American endometrial cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 559.

Transcriptome analysis of an AKT inhibitor-resistant endometrial cancer cell line.

Drug resistance in endometrial cancer (EC) is a serious problem and a barrier to improving prognosis. The PI3K/AKT/mTOR pathway is highly activated in EC and can serve as a potential therapeutic target. Inhibitors against AKT have been developed, but resistance to these inhibitors is a concern. This study aimed to establish AKT inhibitor resistant cell lines and identify differentially expressed genes (DEGs) between parental and AKT inhibitor resistant cell lines to understand the mechanism of drug resistance to AKT inhibitors in EC. The sensitivity of eight EC cell lines to AKT inhibitor was analyzed. One of them was used to establish a drug-resistant cell line. DEGs were examined using RNA sequencing (RNA-seq). Furthermore, DEGs were comprehensively analyzed to identify hub genes. Hub genes were evaluated using quantitative real-time polymerase chain reaction. RNA-seq identified 617 DEGs. Hub genes were selected using bioinformatics analysis. The top 10 hub genes were TNF, CDH1, CCND1, COL1A1, CDH2, ICAM1, CAV1, THBS1, NCAM1, and CDKN2A. Relative mRNA expression was significantly upregulated for TNF, CDH1, CCND1, THBS1, p16INK4a, and p14ARF and significantly downregulated for CDH2, ICAM1, and NCAM1 in borussertib-resistant EC cell line. Drug resistance to AKT inhibitors may depend on genes related to cell adhesion-mediated resistance and transforming growth factor β signaling.

Linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cells and a transgenic model of endometrial cancer

ABSTRACT Emerging evidence has provided considerable insights into the integral function of reprogramming fatty acid metabolism in the carcinogenesis and progression of endometrial cancer. Linoleic acid, an essential fatty acid with the highest consumption in the Western diet regimen, has shown pro-tumorigenic or anti-tumorigenic effects on tumor cell growth and invasion in multiple types of cancer. However, the biological role of linoleic acid in endometrial cancer remains unclear. In the present study, we aimed to investigate the functional impact of linoleic acid on cell proliferation, invasion, and tumor growth in endometrial cancer cells and in a transgenic mouse model of endometrial cancer. The results showed that Linoleic acid significantly inhibited the proliferation of endometrial cancer cells in a dose-dependent manner. The treatment of HEC-1A and KLE cells with linoleic acid effectively increased intracellular reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, caused cell cycle G1 arrest, and induced intrinsic and extrinsic apoptosis pathways. The anti-invasive ability of linoleic acid was found to be associated with the epithelial-mesenchymal transition process in both cell lines, including the decreased expression of N-cadherin, snail, and vimentin. Furthermore, treatment of Lkb1 fl/fl p53 fl/fl transgenic mice with linoleic acid for four weeks significantly reduced the growth of endometrial tumors and decreased the expression of VEGF, vimentin, Ki67, and cyclin D1 in tumor tissues. Our findings demonstrate that linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cell lines and the Lkb1 fl/fl p53 fl/fl mouse model of endometrial cancer, thus providing a pre-clinical basis for future dietary interventions with linoleic acid in endometrial cancer.

The Efficacy of Ganoderma lucidum Extracts on Treating Endometrial Cancer: A Network Pharmacology Approach.

Ganoderma lucidum (GL) is a prominent medicinal mushroom in traditional Chinese medicine, known for its potent antitumor properties. This study aimed to illustrate the efficacy of GL extracts (GLE) on treating endometrial cancer (EC) and explore the underlying mechanisms via network pharmacology and experimental validation. Network pharmacological analysis was conducted to explore the therapeutic efficacy and mechanisms of GL on EC. In vitro experimental validation was performed on human endometrial cancer cell lines HEC-1-A and KLE. Network pharmacology revealed that key targets of GL against EC were primarily associated with the Rap1 signaling pathway. In in vitro experiments, GLE or GGTI-298 (a GTPase inhibitor) treatment inhibited cell proliferation and migration, promoted cell apoptosis, increased caspase-3 level, and arrested cell cycle in G1 phase in HEC-1-A and KLE cells. GLE increased the protein expression of Rap1-GTP, p-AKT, and p-ERK2 in HEC-1-A and KLE cells. Moreover, GGTI-298 enhanced the effects of GLE on suppressing the malignant progression of EC cells and on activating Rap1 signaling pathway. GLE inhibited the malignant progression of EC cells probably via activating the Rap1 signaling pathway.

Abstract B030: PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes

Abstract Cancer cells with DNA repair defects (e.g., BRCA1/2 mutant cells) are vulnerable to PARP inhibitors (PARPi) due to induction of synthetic lethality. However, recent clinical evidence has shown that PARPi can prevent the growth of some cancers irrespective of their BRCA1/2 status, suggesting alternative mechanisms of action. One such mechanism involves DDX21, an RNA helicase that localizes to the nucleoli of cells. When ADP-ribosylated by PARP-1, DDX21 promotes rDNA transcription leading to ribosome biogenesis, protein translation, and ultimately the growth of breast cancer cells. Validating this pathway in other tumor types may lead to the expanded use of PARPi as a cancer therapeutic and the potential use of DDX21 nucleolar localization as a biomarker of clinical response to PARPi. For our first aim, we hypothesized that PARPi also blocks the DDX21/PARP-1/ribosome biogenesis pathway in ovarian and endometrial cancers and can thus be used to inhibit their growth. To test this, we are determining the effect of Niraparib, an inhibitor targeting PARP-1 activity, on DDX21 subcellular localization, ribosome biogenesis, and cell growth in different ovarian (OVCAR3, OVCAR4, and HCC5012) and endometrial (KLE, Ishikawa, and HEC-1-A) cancer cell lines. We are using Western blotting to assess the levels of DDX21 and ADP-ribosylation, and immunofluorescent staining to assess the localization of DDX21 to nucleoli before and after treatment with Niraparib. We are also using qPCR to determine the levels of rRNAs and Puromycin incorporation assays to monitor ribosome translation activity before and after treatment with Niraparib. Moreover, we are using cell growth assays to determine the effect of Niraparib on the proliferation of the ovarian and endometrial cancer cell lines. Our cell-based results indicate that the nucleolar localization of DDX21 is disrupted by Niraparib treatment, leading to a pan-nuclear distribution. Furthermore, Niraparib treatment in ovarian cancer cell lines causes decreased rRNA transcription, decreased protein translation, and decreased cell proliferation. For our second aim, we are assessing possible associations among DDX21 nucleolar localization, PARP-1 protein levels, and clinical outcomes in cancer samples from patients using immunohistochemical (IHC) staining for DDX21 and PARP-1. Analysis of endometrial cancer patient samples suggests a significant association among high DDX21 nucleolar localization, high PARP-1 expression, and decreased survival. Overall, our results suggest that Niraparib works, in part, by blocking the DDX21/PARP-1/ribosome biogenesis pathway leading to endometrial and ovarian cancer cell death. The potential use of DDX21 localization as a biomarker of response to PARPi and as a prognostic factor will be further explored by conducting additional laboratory and clinical studies. Citation Format: Marwa W. Aljardali, Kevin M. Kremer, Jessica Parker, Jayanthi S. Lea, W. Lee Kraus, Cristel V. Camacho. PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes [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 B030.

Uterine Commensal Peptostreptococcus Species Contribute to IDO1 Induction in Endometrial Cancer via Indoleacrylic Acid.

Microbial dysbiosis has an increasingly appreciated impact on carcinogenesis, and the cervicovaginal microbiome plays a critical role in microenvironmental inflammation. Here, we investigated the involvement of the female genital tract Peptostreptococcus species in gynecological cancer via indoleacrylic acid (IAA). IAA production from Peptostreptococcus species and the effect of bacterial culture on tumor growth in vivo were examined. The impact of IAA on cytokine production and indoleamine-2,3-dioxygenase 1 (IDO1) expression in an endometrial cancer (EC) cell line, as well as their effect on Treg and Teff cells, and M1 and M2 macrophage populations were examined in EC patients and tumor-grafted mice. Clinically, Peptostreptococcus species abundance, IAA, and IDO1 expression were verified in EC patients. The results showed that IAA production was induced in the uteri of BALB/c nude mice by Peptostreptococcus species transplantation, and the intratumoral injection of a conditioned medium from Peptostreptococcus cultures into tumor-grafted mice promoted tumor growth. IL-10 expression was upregulated by IAA; IFN-γ expression was increased by IL-10. IFN-γ induced IDO1 expression in the EC cell line. The co-culture of IDO1-expressing EC cells with peripheral blood mononuclear cells upregulated the Treg proportion and decreased the M1/M2 ratio. Clinically, P. anaerobius was more abundant amongst the uterine microbiota of EC patients than the control. The IAA, IDO1, and kynurenine/tryptophan ratios were all higher in EC tissue, and the M1/M2 ratio was lower. Our study sheds light on the link between IDO1 induction and uterine Peptostreptococcus dysbiosis and provides a potential rationale for the role of Peptostreptococcus species in immune tolerance induction in type I endometrial cancer.

Abstract PR003: The combination of the glucagon like peptide-1 receptor agonist semaglutide and the progestin levonorgestrel is highly effective in preclinical studies of endometrial cancer

Abstract Obesity is a major risk factor for endometrial cancer (EC), and glucagon like peptide-1 receptor (GLP-1R) agonists such as semaglutide may be helpful to achieve weight loss during conservative treatment or for EC prevention. Progestins such as levonorgestrel are effective in preventing or treating early-stage EC. However, better and more durable response rates could be achieved using combinatorial therapeutic regimens of well tolerated agents with the potential to enhance progestin effectiveness. We theorized that the combination of semaglutide and levonorgestrel would be useful as a novel treatment or prevention regimen and tested this hypothesis using EC cell lines, patient-derived organoids, and RNA-sequencing from patient samples. From the molecular perspective, EC cell lines, organoids, and tissues express GLP-1R as determined by both qPCR and Western blotting, and GLP-1R agonist treatment induces GLP-1R mRNA transcription through positive feedback mechanisms in EC cell models. Preclinical studies in 6 patient-derived organoid models of EC demonstrated the promising therapeutic effects of the combination of semaglutide and levonorgestrel on endometrial cell viability. Specifically, patient-derived organoids from grade 1 endometrial carcinomas were treated with progesterone, levonorgestrel, semaglutide, or levonorgestrel + semaglutide at drug concentrations of 100 nM for 72 hours. Multiple models demonstrated a significant reduction in viability in response to combinatorial treatment. Most interesting was the induction of not only the membrane GLP-1 receptor with treatment, but also the significant upregulation of nuclear and membrane progesterone receptors, PR and PGRMC1/2, respectively, indicating a positive feedback loop between semaglutide and progestins such as levonorgestrel. In addition, other steroid hormone receptor mRNAs such as the estrogen, androgen and mineralocorticoid receptors (ER, AR, and MR) were similarly upregulated. In these studies, we identify synergistic molecular crosstalk between the GLP-1R and steroid hormone receptor pathways with the potential to enhance the anti-cancer activity of semaglutide and levonorgestrel when combined. Future studies are aimed at testing this regimen in patients conservatively managed for atypical endometrial hyperplasia or low-grade endometrial cancers. Citation Format: Kimberly K. Leslie, Andrea Hagemann, Kristina Thiel, Ian Hagemann, David Mutch, Eric Devor, Paige Malmrose. The combination of the glucagon like peptide-1 receptor agonist semaglutide and the progestin levonorgestrel is highly effective in preclinical studies of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR003.

Abstract B004: Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions

Abstract The purpose of this work is to test the hypothesis that loss of ARID1A promotes endometrial cancer progression by destabilizing tumor cell adherens junctions proteins. ARID1A, an important structural subunit of the SWI/SNF chromatin remodeling complex, is mutated in 26-39% of endometrioid tumors. In endometrial cancer loss of ARID1A has been classically linked to loss of ARID1A expression and has been shown to correlate with increasing tumor grade. However, little is known about the functional impact of ARID1A loss on endometrial cancer progression. A common hallmark of aggressive endometrial cancer is decreased membranous expression of adherens junction proteins, E-Cadherin and β-Catenin. In colorectal and gastric cancer loss of ARID1A expression has been shown to directly downregulate E-Cadherin expression. To determine how loss of ARID1A affects adherens junctions and its impact on endometrial cancer progression we utilized human endometrial cancers and human endometrial cancer cell lines. Using immunohistochemistry, we determined the expression and localization of ARID1A and the adherens junction protein β-Catenin in forty-six human endometrioid type endometrial cancers. Intratumoral heterogeneity of ARID1A expression allowed for quantification of membranous E-Cadherin in relation to presence or absence of ARID1A protein within the same human endometrial tumor. To quantify membranous E-Cadherin we performed immunofluorescence and used line intensity measurements in intratumoral areas of ARID1A presence and ARID1A absence. To assess effects of ARID1A loss on adherens junction in vitro we used short-hairpin RNA to acutely knockdown ARID1A in the endometrial cancer cell lines Ishikawa and ECC-1. Protein expression of ARID1A and E-Cadherin in ARID1A knockdown cells was assessed by Western Blot. Within our cohort of forty-six human endometrial tumors, 75% of tumors with wild-type ARID1A and 45% of tumors with mutated ARID1A had absence of ARID1A protein expression. Due to the discordance between presence of an ARID1A gene mutation and the expression of ARID1A protein, we hypothesize that there are alternative mechanisms of ARID1A protein regulation in endometrial cancer. Additionally, tumors with loss of ARID1A show a trend towards nuclear localization of β-Catenin and have a significant decrease in membranous E-Cadherin expression (P<0.001). However, acute knockdown of ARID1A in Ishikawa and ECC-1 human endometrial cancer cells does not affect total E-Cadherin protein. This suggests that loss of ARID1A may decrease adherens junction stability, as decreased membranous expression of E-Cadherin without total protein change is a common phenotype of destabilized adherens junctions. Thus, we hypothesize that loss of ARID1A destabilizes adherens junctions leading to increased endometrial tumor aggression. Citation Format: Savannah E. Wright, Isurika Weerasinghe, Russell R. Broaddus, Andrew B. Gladden. Investigating loss of ARID1A protein on endometrial cancer progression through the destabilization of adherens junctions [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B004.