Human 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.

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 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.

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 A016: NXP800, a novel, small molecule GCN2 kinase activator, demonstrates potent single-agent activity in ARID1A and ARID1B-deficient endometrial cancer xenograft models

Abstract Background: NXP800 is a clinical stage, antineoplastic, oral, small molecule GCN2 kinase activator that was discovered in a phenotypic screen for inhibitors of the heat shock factor 1-regulated stress response. In a panel of human carcinoma cell lines NXP800 induced the expression of genes associated with activation of the integrated stress response and demonstrated robust antiproliferative activity, particularly in ARID1a-mutated ovarian carcinoma cells lines. In ARID1a-mutated ovarian carcinoma xenografts, treatment with NXP800 resulted in substantial tumor growth inhibition and tumor regression. NXP800 is currently being investigated in a Phase 1b clinical trial in patients with ARID1a-mutated ovarian carcinoma. Here we describe an in vivo study of NXP800 in ARID1a/ARID1b-deficient endometrial cancer xenografts, supporting the clinical development of NXP800 in this indication. Methods: Animal strain: CD1 Nude mice (nu/nu, Charles River). Human endometrial cancer cell lines: RL95-2 (ARID1a mutated, ATCC) and KLE (ARID1a wildtype, ARID1b mutated, ATCC), SNG-M (ARID1a mutated, Creative Bioarray). Xenograft tumors were generated by subcutaneous implantation on the right lower flank of the thigh at a cell density of 2 × 106 cells/mouse, at 0.1 ml Matrigel dilution volume/injection. Experiment groups: Vehicle, NXP800 (35 mg/kg); treatment: QD on days 0-4, 7-11, 14-18, 21-25, 28-30. Loss of ARID1A protein expression was confirmed by western blots. Results: In the SNG-M model, baseline tumor volume for the vehicle and NXP800 groups were 130.2, and 139.7 mm3, respectively, and on Day 32, average tumor volumes were 420.4, and 85.4 mm3, respectively, representing TGI of 81%. In the RL-95 model, baseline tumor volume for the vehicle and NXP800 groups were 124.5, and 129.9 mm3, respectively, and on Day 32, average tumor volumes were 773.8, and 190.3 mm3, respectively, representing TGI of 76%. In the KLE model, baseline tumor volume for both the vehicle and NXP800 groups were 88.1 mm3, respectively, and on Day 32, average tumor volumes were 216.4, and 18.9 mm3, for the vehicle and NXP800 groups, respectively, representing TGI of 91.3%. Conclusions: ARID1A and ARID1B genes encode the alternate, but obligatory, DNA-targeting subunit of the switch/sucrose non-fermentable (SWI/SNF) complex. ARID1A is the most frequently mutated SWI/SNF subunit across cancer types and is mutated in approximately 35% of endometrial carcinomas. NXP800 demonstrated robust antitumor activity in xenografts of endometrial carcinoma, a serious condition for which new treatment options are needed, including sustained tumor growth inhibition both in ARID1A and ARID1B mutated models, supporting the clinical development of NXP800 in endometrial cancer. Citation Format: Ramez N. Eskander, Bradley J. Monk, Brian M. Slomovitz, Enrique Poradosu, Allison Woods, Shay Shemesh, Paul Clarke, Robert Te Poele, Marissa Powers, Paul Workman, Shannon N. Westin. NXP800, a novel, small molecule GCN2 kinase activator, demonstrates potent single-agent activity in ARID1A and ARID1B-deficient endometrial cancer xenograft models [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 A016.

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.

Abstract B023: The role of ribosomal protein L22 in regulating the immune landscape in mismatch repair deficient endometrial cancers

Abstract The purpose of this study is to identify novel mechanisms of immune evasion in mismatch repair deficient endometrial cancers. Despite having a hypermutated phenotype that is proposed to lead to the accumulation of many neoantigens for immune recognition, our group has found that mismatch repair deficient endometrial cancers characterized by MLH1 protein loss display large variations in the number of tumors infiltrating CD8+ T cells (TILs) across different tumors. To better understand how variations in TILs arise in mismatch repair deficient endometrial cancers, we utilized human endometrial cancer samples from the UNCseq cohort. Immunohistochemistry (IHC) was used to identify expression patterns in proteins of interest across mismatch repair deficient endometrial cancers. To determine how expression patterns of proteins of interest correlate with TILs in our patient samples, Definiens Tissue Studio was utilized to quantify immunofluorescent CD8+ TILs in our mismatch repair deficient endometrial cancer cohort. Although upregulation of programmed death ligand 1 (PD-L1) expression is often cited as an explanation for low TILs, analysis of human data by our group shows that PD-L1 expression does not correlate with TIL numbers in mismatch repair deficient endometrial cancers. Interestingly, The Cancer Genome Atlas (TCGA) data reported that ribosomal protein L22 (RPL22) is mutated in ~50% of mismatch repair deficient endometrial cancers. We analyzed RPL22 by IHC and found that it was lost in 52% of our human mismatch repair deficient endometrial cancer cohort. Loss of various ribosomal proteins can alter translation efficiency of proteins critical for promoting immunosurveillance and the immune response in cancer. In our cohort, mismatch repair deficient endometrial cancers that have lost expression of RPL22 have lower numbers of CD8+ TILs. Preliminary data also show that human endometrial cancer cell lines that do not express RPL22 also do not express beta-2-microglobulin (β2M), a component of the major histocompatibility complex I (MHC-I) molecules that is necessary for neoantigen presentation to CD8+ T cells. Thus, we hypothesize that RPL22 loss in mismatch repair deficient endometrial cancers results in decreased translation of β2M and CD8+ T cell evasion. Moving forward, we speculate that RPL22 could be utilized as a predictive biomarker for immunotherapy response in mismatch repair deficient endometrial cancers. Citation Format: Macy L. Osborne, Hannah Atkins, Andrew B. Gladden, Russell R. Broaddus. The role of ribosomal protein L22 in regulating the immune landscape in mismatch repair deficient endometrial cancers [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 B023.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

Deciphering the Role of Insulin-Like Growth Factor 1 in Endometrial Cancer in Patients With Polycystic Ovary Syndrome: Protocol for a Methodological Approach Using Cell Culture Experiments.

Endometrial cancer (EC) is the most common gynecological cancer in women globally. It is linked to increasing obesity rates and longer life spans. The molecular mechanisms leading to EC are unclear; however, women with polycystic ovary syndrome (PCOS) have a 3- to 5-fold increased EC risk. According to a pilot study conducted in the United Kingdom, insulin-like growth factor-1 (IGF-1) gene and protein were raised in the endometrium and blood of women with EC and PCOS, compared with those without PCOS (controls). Therefore, raised serum IGF-1 levels may contribute to an increased EC risk in women with PCOS, but it is necessary to test this hypothesis since not all studies have demonstrated this association. This study aims to investigate the role of IGF-1 in mediating EC risk in PCOS. This will be achieved by evaluating the proliferative effects of PCOS serum, IGF-1, and IGF-1 antagonist on human endometrial cancer 1-A and 1-B cell lines, with a comparison to controls (using serum from women without PCOS and cell culture media). The study will also identify differentially expressed genes and pathways activated by various treatments. We intend to recruit 20 women with PCOS and 20 women without PCOS for this cross-sectional study. All experiments will be carried out 4 times to ensure consistency. We will perform transcriptomic and phosphoproteomic profiling to identify differentially expressed genes and phosphoproteins between different treatments using RNA sequencing and phosphoproteomics. We will also perform bioinformatics pathway analysis to identify whether any unique collection of genes or phosphoproteins explains increased EC risk in PCOS. The primary outcome measure will be the cell proliferation (growth) difference measured by cell index values. Our protocol stands out due to its unique approach; no previous study has used this approach to investigate the oncogenic effect of serum from women with PCOS. Additionally, no previous study has considered the differential mutations of genes related to the insulin signaling pathway across various types of human EC cell lines and the potential impact of these variations on their experimental findings. Participants are currently being recruited. It is expected that preliminary findings suitable for analysis and publication will be available by the summer of 2024. Although we currently do not have any results to report, sharing our protocol at this stage will aid in research collaboration, provide an opportunity for early feedback, and help reduce duplication of effort by other research groups. The findings of our study will have broader implications. A deeper understanding of the mechanisms underlying the regulation of the IGF system in PCOS and EC will improve our ability to develop effective treatment modalities for EC and will be a vital step toward reducing EC in women globally. DERR1-10.2196/48127.

Explore the alterations of downstream molecular pathways caused by ARID1A mutation/knockout in human endometrial cancer cells.

As one of the most common gynecologic malignancies, endometrial cancer (EC) is driven by multiple genetic alterations that may be targeted for treatments. AT-rich interaction domain 1A (ARID1A) gene mutations were reported as early events in endometrial carcinogenesis. To explore the alterations of downstream molecular pathways caused by ARID1A mutations and the associated therapeutic implications, we edited ARID1A gene in human endometrial cancer cell line Ishikawa using the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Proteins (CRISPR/Cas9) technology. We successfully constructed a stable Ishikawa cell line with a confirmed 10bp deletion on the ARID1A gene, which resulted in a code-shift mutation and gene knockout. Compared with unedited wild-type cells, ARID1A knockout (KO) led to reduced apoptosis, accelerated transformation from G0/G1 to S phase, and enhanced cell proliferation. ARID1A deficiency would reduce the protein levels of p21, caspase 7, and caspase 9 in Ishikawa endometrial cancer cells compared with the wild-type cells. In addition, ARID1A KO resulted in high levels of microsatellite instability (MSI-H). Moreover, transcriptomic analyses showed that ARID1A KO can lead to activated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Furthermore, experimental analyses demonstrated that ARID1A KO cells had reduced expression of genetic instability-associated markers mutL homologue 1 (MLH1) and progesterone receptor B (PR) and increased p-Akt expression. These findings support further exploration of ARID1A as a therapeutic target for EC and provide insight into developing more effective treatments in EC, such as the combinatory use of immune checkpoint inhibitors.

Ipatasertib exhibits anti‑tumorigenic effects and enhances sensitivity to paclitaxel in endometrial cancer invitro and invivo.

Endometrial cancer is the most common gynecologic cancer and one of the only cancers for which incidence and mortality is steadily increasing. Although curable with surgery in the early stages, endometrial cancer presents a significant clinical challenge in the metastatic and recurrent setting with few novel treatment strategies emerging in the past fifty years. Ipatasertib (IPAT) is an orally bioavailable pan‑AKT inhibitor, which targets all three AKT isoforms and has demonstrated anti‑tumor activity in pre‑clinical models, with clinical trials emerging for many cancer types. In the present study, the MTT assay was employed to evaluate the therapeutic efficacy of IPAT or IPAT in combination with paclitaxel (PTX) in endometrial cancer cell lines and primary cultures of endometrial cancer. The effect of IPAT and PTX on the growth of endometrial tumors was evaluated in a transgenic mouse model of endometrial cancer. Apoptosis was assessed using cleaved caspase assays and cellular stress was assessed using ROS, JC1 and tetramethylrhodamine ethyl ester assays. The protein expression levels of markers of apoptosis and cellular stress, and DNA damage were evaluated using western blotting and immunohistochemistry. IPAT significantly inhibited cell proliferation, caused cell cycle G1 phase arrest, and induced cellular stress and mitochondrial apoptosis in a dose dependent manner in human endometrial cancer cell lines. Combined treatment with low doses of IPAT and PTX led to synergistic inhibition of cell proliferation and induction of cleaved caspase 3 activity in the human endometrial cancer cell lines and the primary cultures. Furthermore, IPAT effectively reduced tumor growth, accompanied by decreased protein expression levels of Ki67 and phosphorylation of S6 in the Lkb1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The combination of IPAT and PTX resulted in increased expression of phosphorylated‑H2AX and KIF14, markers of DNA damage and microtubule dysfunction respectively, as compared with IPAT alone, PTX alone or placebo‑treated mice. The results of the present study provide a biological rationale to evaluate IPAT and the combination of IPAT and PTX in future clinical trials for endometrial cancer.

A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer

ABSTRACT Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for cancer patients with homologous recombination (HR) deficient tumors. The imipridone ONC206 is an orally bioavailable dopamine receptor D2 antagonist and mitochondrial protease ClpP agonist that has anti-tumorigenic effects in endometrial cancer via induction of apoptosis, activation of the integrated stress response and modulation of PI3K/AKT signaling. Both PARP inhibitors and imipridones are being evaluated in endometrial cancer clinical trials but have yet to be explored in combination. In this manuscript, we evaluated the effects of the PARP inhibitor olaparib in combination with ONC206 in human endometrioid endometrial cancer cell lines and in a genetically engineered mouse model of endometrial cancer. Our results showed that simultaneous exposure of endometrial cancer cells to olaparib and ONC206 resulted in synergistic anti-proliferative effects and increased cellular stress and apoptosis in both cell lines, compared to either drug alone. The combination treatment also decreased expression of the anti-apoptotic protein Bcl-2 and reduced phosphorylation of AKT and S6, with greater effects compared to either drug alone. In the transgenic model of endometrial cancer, the combination of olaparib and ONC206 resulted in a more significant reduction in tumor weight in obese and lean mice compared to ONC206 alone or olaparib alone, together with a considerably decreased Ki-67 and enhanced H2AX expression in obese and lean mice. These results suggest that this novel dual therapy may be worthy of further exploration in clinical trials.

A ciprofloxacin derivative with four mechanisms of action overcomes paclitaxel resistance in p53-mutant and MDR1 gene-expressing type II human endometrial cancer

Dysfunction of the p53 gene and the presence of the MDR1 gene are associated with many malignant tumors including endometrial cancer and are responsible for cancer therapeutic resistance and poor survival. Thus, there is a critical need to devise novel combinatorial therapies with multiple mechanisms of action to overcome drug resistance. Here, we report a new ciprofloxacin derivative (CIP2b) tested either alone or in combination with taxanes against four human endometrial cancer cell lines. In vitro studies revealed that a combination of paclitaxel + CIP2b had synergistic cytotoxic effects against MDR1-expressing type-II human endometrial cancer cells with loss-of-function p53 (Hec50co LOFp53). Enhanced antitumor effects were confirmed by substantial increases in caspase-3 expression, cell population shifts toward the G2/M phase, and reduction of cdc2 phosphorylation. It was found that CIP2b targets multiple pathways including the inhibition of MDR1, topoisomerase I, and topoisomerase II, as well as enhancing the effects of paclitaxel (PTX) on microtubule assembly. In vivo treatment with the combination of PTX + CIP2b also led to significantly increased accumulation of PTX in tumors (compared to CIP2b alone) and reduction in tumor growth. Enhanced in vivo cytotoxic effects were confirmed by histological and immunohistochemical examination of the tumor tissues. Complete blood count and blood biochemistry data confirmed the absence of any apparent off-target toxicity. Thus, combination therapy involving PTX and CIP2b targeted multiple pathways and represents an approach that could result in improved tolerance and efficacy in patients with type-II endometrial cancer harboring the MDR1 gene and p53 mutations.

The Glutaminase Inhibitor Compound 968 Exhibits Potent In vitro and In vivo Anti-tumor Effects in Endometrial Cancer.

Glutamine is one of the primary nutrients utilized by cancer cells for energy production and biosynthesis. Hence, interfering with glutamine metabolism may impose anti-tumor effects. In this study, we assessed the anti-tumorigenic effects of glutaminase-1 enzyme (GLS1) inhibition in endometrial cancer in vitro and in vivo. The human endometrial cancer cell lines Ishikawa and HEC-1B were used. The effects of compound 968 on cell proliferation, cell cycle, apoptosis, cellular stress, and AKT/mTOR pathway inhibition were assessed. The synergistic effects of compound 968 and paclitaxel were also analyzed. The in vivo effect of compound 968 was evaluated using tumor xenografts. We found that the GLS1-targeting compound 968 was able to reduce cancer cell proliferation in a dose- and time-dependent manner. Compound 968 combined with a low concentration of paclitaxel showed stronger inhibitory effects. Further analyses indicated that compound 968 induced cell cycle arrest at the G1 phase, as well as increased the production of cellular reactive oxygen species (ROS) and promoted cellular stress and cancer cell apoptosis. Additionally, the treatment of endometrial cancer with compound 968 downregulated the expression of GLS1 and cyclin D1 and upregulated the expression of P21 and E-cadherin. Moreover, the treatment of endometrial cancer cells with compound 968 significantly reduced the levels of phospho-S6 ribosomal protein and phospho-AKT (Ser473), indicative of AKT/mTOR/S6 signaling pathway inhibition. In xenograft mouse models of endometrial cancer, compound 968 significantly suppressed tumor growth. In addition, western blotting analysis indicated that GLS1 expression was upregulated in human endometrial cancer tissues. Compound 968 may be a promising approach for the management of human endometrial cancer.

Abstract P015: Mechanisms of mitochondrial dysfunction in development of mismatch repair-deficient endometrial cancer

Abstract Background: Lynch syndrome (LS) is a hereditary cancer syndrome carrying roughly 60% lifetime risk of endometrial cancer (EC) development. LS-EC is caused by mutations in mismatch repair genes (most commonly MSH2), which lead to DNA repair deficiency and hypermutability. Identifying mechanisms that impact cancer development could provide novel opportunities for EC prevention. Our recent studies in Msh2-deficient mice identified mitochondrial dysfunction in EC pathogenesis. The objective of this study is to define the impact of MSH2 loss on mitochondrial function and oxidative DNA damage to identify targets for EC prevention. We hypothesized that MSH2 loss disrupts mitochondrial function and increases oxidative DNA damage in EC cells. Methods: The effects of MSH2 loss on EC pathogenesis were evaluated using our novel mouse model for EC development (PR-Cre+Msh2flox/flox, abbreviated Msh2KO) and primary cell lines derived from this model. Mitochondrial stress tests (MSTs) compared mitochondrial function in murine and human MSH2-deficient and -intact EC cell lines. Western blots compared mitochondrial protein expression between cell lines. Transcriptomic data of human ECs from The Cancer Genome Atlas (TCGA) were probed using cBioPortal. To examine direct effects of MSH2 loss on oxidative stress and mitochondrial function, we performed lentiviral shRNA knockdown (KD) of MSH2 in two human EC cell lines (KLE, Hec1a) and MSH2 overexpression (MSH2++) in MFE280 cells. MSTs and Western blots were utilized as above. Immunofluorescent staining compared oxidative DNA damage in MSH2-KD, MSH2++, and control cell lines after pharmacologically induced oxidative stress via H2O2. RESULTS Transcriptomic signatures of histologically normal endometrium and EC from Msh2KO mice versus control mice reveal mitochondrial dysfunction as a key aberrant pathway. MSTs show reduced baseline and induced mitochondrial function in murine and human MSH2-deficient EC cells compared to MSH2-intact (p<0.0001). Western blots show that MSH2-deficient EC cells exhibit loss of mitochondrial complex II compared to MSH2-intact cells (p<0.05). TCGA data confirm MSH2-deficient human ECs have reduced complex II subunits (p<0.05). Isogenic in vitro studies show MSH2-KD reduces complex II expression yet has variable effects on mitochondrial function. MSH2 overexpression increases complex II expression and mitochondrial function. Oxidative DNA damage did not differ at baseline, but H2O2-induced DNA damage increased after MSH2-KD in one of two cell lines. Conclusions: MSH2 and complex II loss are strongly associated in EC. Further studies on the downstream effects of complex II loss are necessary to determine the contribution to LS-EC development. Complex II loss could result in disruptions to oxidative phosphorylation, the citric acid cycle, metabolic reprogramming, and oncometabolite production. Defining direct effects of MSH2 loss on these potential cancer susceptibility mechanisms will open new avenues for LS-associated EC prevention. Citation Format: Diana L. Moreno, Mikayla S. Borthwick, Leah Peralta, Wai Kin Chan, Brenda Melendez, Qian Zhang, Nisha Gokul, Melinda S. Yates. Mechanisms of mitochondrial dysfunction in development of mismatch repair-deficient endometrial cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P015.

Effects of RFRP‑3 on an ovariectomized estrogen‑primed rat model and HEC‑1A human endometrial carcinoma cells.

The hypothalamic peptide gonadotropin inhibitory hormone (GnIH) is a relatively novel hypothalamic neuropeptide, identified in 2000. It can influence the hypothalamic-pituitary-gonadal axis and reproductive function through various neuroendocrine systems. The present study aimed to explore the effects and potential underlying molecular mechanism of RFamide-related peptide-3 (RFRP-3) injection on the uterine fluid protein profile of ovariectomized estrogen-primed (OEP) rats using proteomics. In addition, the possible effects of RFRP-3 on the viability and apoptosis of the human endometrial cancer cell line HEC-1A and associated molecular mechanism were investigated. The OEP rat model was established through injection with GnIH/RFRP-3 through the lateral ventricle. At 6 h after injection, the protein components of uterine fluid of rats in the experimental and control groups were analyzed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Differentially expressed proteins (DEPs) were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interactions (PPI) were investigated using the STRING database. PPI networks were then established before hub proteins were selected using OmicsBean software. The expression of one of the hub proteins, Kras, was then detected using western blot analysis. Cell Counting Kit-8, Annexin V-FITC/PI, reverse transcription-quantitative PCR and western blotting were also performed to analyze cell viability and apoptosis. In total, 417 DEPs were obtained using LC-MS/MS, including 279 upregulated and 138 downregulated proteins. GO analysis revealed that the majority of the DEPs were secretory proteins. According to KEGG enrichment analysis, the DEPs found were generally involved in tumor-associated pathways. In particular, five hub proteins, namely G protein subunit α (Gna)13, Gnaq, Gnai3, Kras and MMP9, were obtained following PPI network analysis. Western blot analysis showed that expression of the hub protein Kras was downregulated following treatment with 10,000 ng/ml RFRP-3. RFRP-3 treatment (10,000 ng/ml) also suppressed HEC-1A cell viability, induced apoptosis, downregulated Bcl-2 and upregulated Bax protein expression, compared with those in the control group. In addition, compared with those in the control group, RFRP-3 significantly reduced the mRNA expression levels of PI3K, AKT and mTOR, while upregulating those of LC3-II. Compared with those in the control group, RFRP-3 significantly decreased the protein expression levels of PI3K, AKT, mTOR and p62, in addition to decreasing AKT phosphorylation. By contrast, RFRP-3 significantly increased the LC3-II/I ratio and G protein-coupled receptor 147 (GPR147) protein expression. In conclusion, the present data suggest that RFRP-3 can alter the protein expression profile of the uterine fluid of OEP rats by upregulating MMP9 expression whilst downregulating that of key hub proteins Gna13, GnaQ, Gnai3 and Kras. Furthermore, RFRP-3 can inhibit HEC-1A cell viability while promoting apoptosis. The underlying molecular mechanism may involve activation of GPR147 receptor by the direct binding of RFRP-3, which further downregulates the hub protein Kras to switch on the PI3K/AKT/mTOR pathway. This subsequently reduces the Bcl-2 expression and promotes Bax expression to induce autophagy.

Pentamidine inhibits proliferation, migration and invasion in endometrial cancer via the PI3K/AKT signaling pathway

BackgroundPentamidine has been reported to have many pharmacological effects including anti- protozoal, anti-inflammatory, and anti-tumor activities. The aim of this study is to investigate the potential therapeutic role of Pentamidine and molecular mechanisms of Pentamidine on PI3K/AKT signaling pathway underlying the anti-tumor properties in endometrial cancer.MethodsOur study was carried out in the central laboratory of Harbin Medical University from 2019 to 2021. Human endometrial cancer cell lines Ishikawa and HEC-1A were treated with Pentamidine. The proliferation ability of cells was investigated by MTS and colony formation assays. The cell cycle distribution was detected by flow cytometry. Cell migration and invasion were analyzed by using the wound healing assay and Transwell assay. Western blotting was performed to measure the levels of AKT, p-AKT, MMP-2, and MMP-9.ResultsOur results revealed that treatment of Pentamidine inhibited proliferation, migration and invasion of Ishikawa and HEC-1A endometrial cancer cells. Mechanistic investigation showed that Pentamidine inhibited PI3K/AKT signaling pathway and also reduced the expression of MMP-2 and MMP-9. In addition, co-treatment with PI3K kinase inhibitor LY294002 and Pentamidine leaded to increased repression of cell viability and the protein expression of p-AKT in Ishikawa cells.ConclusionsPentamidine suppresses PI3K/AKT signaling pathway, and inhibits proliferation, migration and invasion of EC cells. These findings suggested that Pentamidine might be a potential candidate for treating EC through PI3K/AKT pathway.

Effects of glabridin on cell proliferation and long non-coding RNA expression in HEC-1B cells

Purpose: Endometrial cancer is one of the most common gynecological cancers in the world. Glabridin is a main isoflavone in Glycyrrhiza glabra (licorice) root. It has several therapeutic effects such as anti-proliferative and anti-inflammatory. Long non-coding RNAs (LncRNAs) play a role in a variety of cellular processes, and their abnormal expression may contribute to tumor development and progression. In this study, the effects of glabridin on LncRNAs gene expression and viability of HEC-1B human endometrial cancer cell lines have been investigated. 
 Materials and methods: Glabridin was applied to HEC-1B cells in concentrations of 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM. Glabridin's effect on HEC-1B cell proliferation was also evaluated using MTS assay. Expression profiles of LncRNAs such as H19, RNU43, LNC-MYC-3:1 and ABCC5-AS1:1 were determined by real-time PCR. 
 Results: Glabridin reduced the viability of HEC-1B cells in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) dose in HEC-1B cells was detected to be 21.32 μM and 13.5 μM at the 24th and 48 hours, respectively. Glabridin has been observed to cause a significant decrease in the expression of H19 and RNU43 while increasing in the expression of LNC-MYC-3:1 and ABCC5-AS1:1.
 Conclusion: Glabridin could induce HEC-1B cell death by regulating LncRNAs expression. As a result, glabridin is a potential candidate for a more effective therapeutic agent against human endometrial cancer.

Lidocaine inhibits the proliferation and migration of endometrial cancer cells, and promotes apoptosis by inducing autophagy.

As a gynecological malignancy, endometrial cancer (EC) has a high incidence and mortality rate in women. The aim of the present study was to investigate the mechanism of EC and to identify novel effective treatment methods for this disease. The viability and proliferation of the RL95-2 human endometrial cancer cell line were assessed using Cell Counting Kit-8 assays. Colony formation, wound healing, Transwell, TUNEL and immunofluorescence assays were used to assess the effects of 5, 10 and 15 mM lidocaine on the colony formation, migration, invasiveness, apoptosis and Beclin 1 protein expression of RL95-2 cells, respectively. Furthermore, western blotting was used to analyze the protein expression levels of apoptosis- and autophagy-related proteins. The results demonstrated that lidocaine inhibited the viability, proliferation and migration of EC cells, and promoted apoptosis. Furthermore, lidocaine was demonstrated to induce autophagy and Beclin 1 protein expression in EC cells. In conclusion, lidocaine inhibited the proliferation and migration of EC cells, and promoted apoptosis via autophagy induction, which indicated that lidocaine may be a potential therapeutic drug for the treatment of EC.

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)