Pathogenesis Of Endometrial Cancer Research Articles

The Possible Role of Mycotoxins in the Pathogenesis of Endometrial Cancer

Endometrial cancer is one of the most common cancer types among women. Many factors can contribute to the development of this disease, including environmental factors and, thus, eating habits. Our study aims to determine the levels of various mycotoxins and their metabolites in the blood serum and endometrial tissue samples of participants with previously proven endometrial cancer and to find possible contributions to cancer development. In the cohort clinical trial, 52 participants aged between 44 and 86 were studied. The participants were divided into two groups: patients or matched controls. All patients had previously histologically diagnosed endometrial cancer. The cancer patients were divided into low-grade endometrioid and low- plus high-grade endometrioid groups. Controls had no history of endometrial malignancy or premalignancy. Blood serum and endometrial tissue samples were obtained from all study patients. We compared the concentrations of total Aflatoxins (Afs), Deoxynivalenol (DON), Ochratoxin-A (OTA), T2-toxin and HT2 toxin (T2/HT2 toxin), Zearalenone (ZEN), alpha-Zearalenol (α-ZOL), and Fumonisin B1 (FB1) in the serum and endometrium between the different study groups. As a result, we can see a significant correlation between the higher levels of Afs and zearalenone and the presence of endometrial cancer. In the case of Afs, DON, OTA, T2/HT2 toxins, ZEN, and alpha-ZOL, we measured higher endometrial concentrations than in serum. Considering the effect of mycotoxins and eating habits on cancer development, our results might lead to further research exploring the relationship between certain mycotoxins and endometrium cancer.

Establishing and Validating an Innovative Focal Adhesion-Linked Gene Signature for Enhanced Prognostic Assessment in Endometrial Cancer.

Studies have highlighted the significant role of focal adhesion signaling in cancer. Nevertheless, its specific involvement in the pathogenesis of endometrial cancer and its clinical significance remains uncertain. We analyzed TCGA-UCEC and GSE119041 datasets with corresponding clinical data to investigate focal adhesion-related gene expression and their clinical significance. A signature, "FA-riskScore," was developed using LASSO regression in the TCGA cohort and validated in the GSE dataset. The FA-riskScore was compared with four existing models in terms of their prediction performance. We employed univariate and multivariate Cox regression analyses towards FA-riskScore to assess its independent prognostic value. A prognostic evaluation nomogram based on our model and clinical indexes was established subsequently. Biological and immune differences between high- and low-risk groups were explored through functional enrichment, PPI network analysis, mutation mining, TME evaluation, and single-cell analysis. Sensitivity tests on commonly targeted drugs were performed on both groups, and Connectivity MAP identified potentially effective molecules for high-risk patients. qRT-PCR validated the expressions of FA-riskScore genes. FA-riskScore, based on FN1, RELN, PARVG, and PTEN, indicated a poorer prognosis for high-risk patients. Compared with published models, FA-riskScore achieved better and more stable performance. High-risk groups exhibited a more challenging TME and suppressive immune status. qRT-PCR showed differential expression in FN1, RELN, and PTEN. Connectivity MAP analysis suggested that BU-239, potassium-canrenoate, and tubocurarine are effective for high-risk patients. This study introduces a novel prognostic model for endometrial cancer and offers insights into focal adhesion's role in cancer pathogenesis.

Abstract 3377: Exploring the role of obesity-induced exosome secretion and associated proteins in endometrial cancer pathogenesis: A roadmap for prevention through exosome inhibition

Abstract Introduction: Endometrial cancer (EC) is the leading gynecologic malignancy in the U.S., with obesity implicated in 57% of cases. This study explores the molecular workings of oncogenic protein expression (TMEM205, STAT5, and FAS) and their role in regulating exosome secretion. Understanding these mechanisms is crucial for unraveling pathways involved in obesity-related EC, informing the development of innovative strategies for prevention and treatment. Methods: The isolated exosomes were quantified using nanoparticle tracking analyzer (NTA) and their size measured by Transmission electron microscopy (TEM). TMEM205, STAT5, FAS and PIAS3 expression was confirmed by IHC, ELISA and RT-PCR in patient and high fat diet treated mouse tissue. Endometrial hyperplasia was developed in immunocompetent mice using high fat diet (HFD; 45 kcal% fat diet) for 16 weeks. Results: Our study investigates exosome secretion and the regulation of oncogenic proteins in adipose and uterine tissues from obese EC patients compared to non-obese EC samples. Analyzing the effects of a 45% kcal high-fat diet (HFD) on mice over 24 weeks, we observed higher body weight and increased adipose tissue in the HFD group, along with enlarged uterine horns and heightened inflammation. This correlated with elevated exosome secretion and increased expression of TMEM205, FAS and STAT5, while the tumor suppressor gene PIAS3 was downregulated. The identified small molecule inhibitor, DAP-5, selectively targeting TMEM205 and exosome secretion, demonstrated a significant reduction in body weight and adipose tissue accumulation when administered to HFD mice. Additionally, DAP-5 treatment restored normal uterine morphology and reduced the expression of exosome-related proteins. Conclusion: This study offers central insights into the mechanisms underlying obesity-mediated TMEM205 expression and exosome secretion, shedding light on their role in the pathogenesis of EC. Additionally, it provides pre-clinical evidence supporting the initiation of the first in-human studies for exosome-targeted therapies aimed at preventing obesity-mediated EC. Citation Format: Takahiko Sakaue, Kalpana Deepa Priya Dorayappan, Roman Zingarelli, Wafa Khadraoui, Muralidharan Anbalagan, John Wallbillich, Mark Wascovich, Pragyan Paramita, Adrian Suarez, Casey Cosgrove, Larry G. Maxwell, Hironori Koga, David O’Malley, David Cohn, Selvendiran Karuppaiyah. Exploring the role of obesity-induced exosome secretion and associated proteins in endometrial cancer pathogenesis: A roadmap for prevention through exosome inhibition [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 3377.

Abstract A010: Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis

Abstract Background: Lynch syndrome (LS), defined by mutations in DNA mismatch repair genes including MSH2, carries a 60% lifetime risk of developing endometrial cancer (EC). Our recent studies in Msh2-deficient mice revealed mitochondrial dysfunction in EC pathogenesis. We aimed to define mechanisms of mitochondrial dysfunction and impact on metabolism to identify targets for EC prevention. Methods: We assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines from this model, and human EC cells with isogenic MSH2-knockdown. mtDNA damage was measured using RT-PCR. Mitochondrial content was quantified in vitro by immunofluorescence (IF) for TOM20 and in vivo by anti-TOM20 immunohistochemistry (IHC). Mitochondrial stress tests (MSTs) assessed mitochondrial function in vitro. Metabolite profiling via high resolution ion chromatography mass spectrometry was conducted on Msh2KO EC and MSH2-intact EC from the Pten +/− mouse model. Glycolysis was suppressed in vitro by replacing glucose with galactose in culture media. Results: Baseline mtDNA damage was elevated in MSH2-deficient MFE280 and RL95-2 EC cell lines by 4.8- to 7.9-fold compared to MSH2-intact KLE and Hec50 cells (p<0.001). MSH2 knockdown increased mtDNA damage in KLE and Hec50 cells by 2.5- to 5.7-fold (p<0.05). MSH2-deficient mouse and human cell lines had lower mitochondrial content compared to MSH2-intact counterparts as indicated by TOM20 staining, with over 60% reductions in mitochondrial volume in MSH2-deficient mouse cell lines and over 95% reductions in MSH2-deficient human EC cells compared to respective controls (p<0.0001). IHC showed reduced mitochondrial content during EC development in Msh2KO mice and in human LS EC tissues. MSTs revealed decreased baseline and induced mitochondrial function in Msh2KO cells relative to controls and in MSH2-deficient human cells (MFE280, RL95-2, and Hec59) compared to MSH2-intact human cells (KLE, Hec1a, Hec50), with p<0.0001. Within the top 10 differentially modulated metabolites in Msh2KO EC compared to Pten +/− EC were glucose 6-phosphate (log2 fold-change (FC) -1.79), mannose 6-phosphate (log2FC -1.77), fructose 6-phosphate (log2FC -1.54), and lactate (log2FC 0.68), padj<0.05. Galactose reduced viability in MSH2-deficient mouse and human cells more than in their MSH2-intact counterparts (p<0.01). Conclusions: We identified mitochondrial depletion as a novel phenotype of MSH2-deficient EC. mtDNA damage in MSH2-deficient cells suggests a role for MSH2 in mtDNA integrity. MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. Decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis, signified by increased usage of glycolytic intermediates and dependence on glycolysis for survival. Mitochondrial and metabolic aberrations could be evaluated as novel biomarkers for EC development and targets for prevention in women with LS. Citation Format: Mikayla B. Bowen, Brenda Melendez, Anna Zal, Collene Jeter, Wai Kin Chan, Lin Tan, Philip Lorenzi, Diana Moreno, Leah Peralta, Qian Zhang, Nisha Gokul, Karen Lu, Hyun-Eui Kim, Melinda S. Yates. Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis [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 A010.

Abstract IA013: Unique metabolic changes in Lynch syndrome-associated endometrial cancer

Abstract Lynch syndrome-associated endometrial cancers (ECs) are defined by germline mutations in DNA mismatch repair genes, including MSH2, MSH6, PMS2, and MLH1. Lynch syndrome (LS) tumors have received increased attention in the therapeutic context as these tumors are microsatellite instable and often reflect increased immune cell infiltration and enhanced response to immunotherapy. However, relatively little attention has been paid to pathogenesis of LS ECs. Women with LS represent an at-risk population who could benefit from cancer interception strategies due to a 60% cumulative lifetime risk of developing EC. We developed a mouse model of MSH2 loss targeted to the uterus to evaluate EC pathogenesis, with the goal of elucidating mechanisms that alter disease penetrance for women with hereditary risk for EC development. Our Progesterone Receptor-Cre Msh2f lox/flox mice (abbreviated Msh2KO) develop ECs that closely mimic human EC. Whole transcriptome profiling of Msh2KO EC and Msh2KO normal endometrium compared to wild-type endometrium identified “Mitochondrial dysfunction” as a significantly altered pathway in Msh2 deficiency, prompting our in-depth evaluation of mitochondrial alterations in Msh2KO mice, MSH2-deficient cell lines, and LS-associated EC patient specimens. We will discuss the impact of MSH2 deficiency across multiple measures of mitochondrial content, integrity, function, and metabolomic analyses in ECs using preclinical models and human tissue studies. Overall, our studies reveal that MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. In addition, decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis. We are conducting further studies to define mechanisms underlying mitochondrial defects due to MSH2 loss and opportunities for intervention. Mitochondrial and metabolic aberrations could serve as novel biomarkers for EC development and targets for cancer prevention in women with LS. Citation Format: Melinda S. Yates. Unique metabolic changes in Lynch syndrome-associated 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 IA013.

Abstract IA012: The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules

Abstract Uterine cancer is the most common gynecologic malignancy in the United States with approximately 60,000 new diagnoses each year. While many cases of uterine endometrial carcinoma (EMCA) have favorable outcomes with recurrence-free long-term survival, outcomes for the high-grade, treatment-refractory histological subtypes, including uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS), remain an important clinical problem. Uterine serous carcinomas and uterine carcinosarcomas tumors are the most aggressive subtypes of uterine cancer, displaying a high propensity to invade the peritoneal cavity and to metastasize to distant sites. Accordingly, 50-70% of high-grade tumors exhibit extrauterine spread at time of initial surgery and have limited treatment options. The inherent aggressiveness of these tumors suggests that they are intrinsically equipped to disseminate and propagate tumors at secondary sites. The molecular drivers of this phenotype, however, have yet to be completely characterized. A lack of established and validated molecular drivers of the high-grade subtypes, especially those that are responsible for the high rate of metastasis, has limited the use of targeted treatment strategies. However, we and other groups have shown that PPP2R1A, the scaffolding subunit of the tumor suppressor protein phosphatase 2A, is mutated in up to 40% of USC patients, and these recurrent mutations are found at both the primary and metastatic sites. These mutations are heterozygous, and two specific mutations: P179R and S256F, occur exclusively within these high-grade subtypes of uterine cancer (i.e., are not found in any other cancer type). PP2A, is a family of serine/threonine phosphatases, where the active heterotrimeric form of the enzyme is comprised of a catalytic (C) subunit, a scaffolding (A) subunit, and a substrate directing regulatory (B) subunit. In aggregate, PP2A is considered a tumor suppressor protein, but certain heterotrimers have tumor promoting roles. Here we present data related to the molecular and biological basis through which these recurrent pathogenic mutations drive endometrial cancer development and progression using disease relevant cellular and in vivo model systems. Additionally, building from previously published work from our group, we have developed a novel series of small molecule molecular glues (PMGs) that specifically correct the mutant heterotrimer driving tumor regressions in both cellular and patient derived xenograft models of high-grade endometrial cancer. Collectively, this data suggests that recurrent pathogenic mutations in the PPP2R1A gene drive endometrial cancer pathogenesis and these mutations can be therapeutically targeted using a series of pharmaceutically tractable small molecules. Citation Format: Goutham Narla. The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules [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 IA012.

Abstract IA011: Understanding field carcinogenesis in obesity-related endometrial cancer

Abstract Over the last decade, endometrial cancer (EC) incidence has increased at an average rate of 0.8% each year. Although cancer death rates decreased during this time, EC showed the greatest increase in cancer death among women. Public awareness of EC remains low, and EC research remains underfunded relative to its societal burden. While most women with EC are post-menopausal, up to 30% of cases occur in pre-menopausal women under 50 years of age, with obesity being a major risk factor. More than half of all EC is associated with obesity. Obesity is also a leading risk factor for benign endometrial hyperplasia, which is considered the precursor lesion to EC. Like other cancers, the causes of EC are multifactorial, with obesity and somatic mutations playing causal roles. By 2030, half of all adult women in the U.S. are expected to be classified as obese. Therefore, EC incidence is expected to increase. If detected early, EC can be cured with surgical hysterectomy, but this solution is not ideal for all women. Intentional weight loss can lower endometrial cancer risk in obese women, but certain barriers prevent obese, at-risk women from losing weight or maintaining healthy lifestyles while living in obesogenic environments. There is a critical need to understand the biological mechanisms underlying EC pathogenesis in at-risk obese women. Although EC is thought to arise from a hyperplastic endometrial epithelium, our previous findings suggest that high-fat diet induced obesity affects other cell types within the endometrial microenvironment, including endometrial stroma and immune cells, in the absence of histopathological change. Common EC somatic mutations can also arise in the absence of histopathological change or occur in cases of benign disease. Our overarching hypothesis is that a normal appearing endometrium may accrue cell-context dependent alterations in response to obesity and somatic mutations across a pre-malignant tissue field that are predictive of malignant transformation. This presentation will highlight our recent work on the combined molecular and phenotypic effects of obesity and common EC driver mutations on the endometrial tissue field. The identification of high-value molecular targets across the endometrial pre-malignant field may inform new early detection and prevention strategies for EC. Citation Format: Ronald Chandler. Understanding field carcinogenesis in obesity-related 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 IA011.

Transcription Factor STAT3-Activated LDHB Promotes Tumor Properties of Endometrial Cancer Cells by Inducing MDH2 Expression.

The pathogenesis of endometrial cancer (EC) involves the regulation of lactate dehydrogenases. However, the role and mechanism of lactate dehydrogenase-B (LDHB) in EC progression have not been studied. The mRNA levels of LDHB and malate dehydrogenase 2 (MDH2) were detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blotting and immunohistochemistry assays. Cell proliferation, apoptosis, and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine, transwell, and flow cytometry assay, respectively. Glycolysis was investigated using Glucose Assay Kit, CheKine™ Micro Lactate Assay Kit, and ADP/ATP ratio assay kit. An in vivo tumor formation assay was conducted to disclose the effect of LDHB on tumor growth in vivo. The associations among signal transducer and activator of transcription 3 (STAT3), LDHB, and MDH2 were predicted through JASPAR or GeneMANIA online database and identified by chromatin immunoprecipitation assay, dual-luciferase reporter assay, and co-immunoprecipitation assay. LDHB expression was increased in EC tissues and cells in comparison with normal endometrial tissues and human endometrial stromal cells. LDHB had the potential as a biomarker to predict the prognosis of EC patients. In addition, LDHB knockdown inhibited the proliferation, invasion, and glycolysis and promoted apoptosis of RL95-2 and Ishikawa cells. LDHB knockdown inhibited tumor property of Ishikawa cells in vivo. STAT3 bound to the promoter region of LDHB, and STAT3 silencing-induced effects were relieved after LDHB upregulation. LDHB interacted with and regulated MDH2 expression. Moreover, MDH2 overexpression rescued LDHB knockdown-induced effects on EC cell phenotypes. STAT3-activated LDHB promoted endometrial cancer cell malignancy by inducing MDH2 production.

ACSL1-Mediated Fatty Acid β-Oxidation Enhances Metastasis and Proliferation in Endometrial Cancer.

Gynecological malignancies, such as endometrial cancer (EC) and uterine cancer are prevalent. Increased Acyl-CoA synthetase long-chain family member 1 (ACSL1) activity may contribute to aberrant lipid metabolism, which is a potential factor that contributes to the pathogenesis of endometrial cancer. This study aimed to elucidate the potential molecular mechanisms by which ACSL1 is involved in lipid metabolism in endometrial cancer, providing valuable insights for targeted therapeutic strategies. Xenograft mouse models were used to assess the effect of ACSL1 on the regulation of endometrial cancer progression. ACSL1 protein levels were assessed via immunohistochemistry and immunoblotting analysis. To assess the migratory potential of Ishikawa cells, wound-healing and Transwell invasion assays were performed. Changes in lipids in serum samples from mice with endometrial cancer xenotransplants were examined in an untargeted lipidomic study that combined multivariate statistical methods with liquid chromatography‒mass spectrometry (LC/MS). Patient sample and tissue microarray data suggested that higher ACSL1 expression is strongly associated with the malignant progression of EC. Overexpression of ACSL1 enhances fatty acid β-oxidation and 5'-adenylate triphosphate (ATP) generation in EC cells, promoting cell proliferation and migration. Lipidomic analysis revealed that significant changes were induced by ACSL1, including changes to 28 subclasses of lipids and a total of 24,332 distinct lipids that were detected in both positive and negative ion modes. Moreover, pathway analysis revealed the predominant association of these lipid modifications with the AMPK/CPT1C/ATP pathway and fatty acid β-oxidation. This study indicates that ACSL1 regulates the AMPK/CPT1C/ATP pathway, which induces fatty acid β-oxidation, promotes proliferation and migration, and then leads to the malignant progression of EC.

The ErbB Signaling Network and Its Potential Role in Endometrial Cancer.

Endometrial cancer (EC) is the second most common malignancy of the female reproductive system worldwide. The updated EC classification emphasizes the significant role of various signaling pathways such as PIK3CA-PIK3R1-PTEN and RTK/RAS/β-catenin in EC pathogenesis. Some of these pathways are part of the EGF system signaling network, which becomes hyperactivated by various mechanisms and participates in cancer pathogenesis. In EC, the expression of ErbB receptors is significantly different, compared with the premenopausal and postmenopausal endometrium, mainly because of the increased transcriptional activity of ErbB encoding genes in EC cells. Moreover, there are some differences in ErbB-2 receptor profile among EC subgroups that could be explained by the alterations in pathophysiology and clinical behavior of various EC histologic subtypes. The fact that ErbB-2 receptor expression is more common in aggressive EC histologic subtypes (papillary serous and clear cell) could indicate a future role of ErbB-targeted therapies in well-defined EC subgroups with overexpression of ErbB receptors.

A novel epithelial-mesenchymal transition-related lncRNA signature predicts prognosis and immune status in endometrioid endometrial cancer.

The pathogenesis and progression of endometrial cancer (EC) are associated with epithelial-mesenchymal transition (EMT) and long noncoding RNAs (lncRNAs). In the present study, we aimed to identify an EMT-related lncRNA signature and evaluate its prognostic value in EC. We obtained the expression profile of lncRNAs and clinical information of patients with endometrioid EC from The Cancer Genome Atlas database (N = 401). We identified a signature of 5 EMT-related lncRNAs and calculated the risk score of each patient. Next, we validated the independence of the prognostic value of the EMT-related lncRNA signature. Furthermore, we performed Gene Set Enrichment Analysis to identify potential molecular function and Kyoto Encyclopedia of Genes and Genomes pathways related to the EMT-related lncRNA signature. Tumor microenvironment analysis and immune checkpoint blockade (ICB) response prediction were also assessed. Survival analysis revealed that the high-risk group, based on the EMT-related lncRNA signature, had a poorer prognosis than the low-risk group in the training, testing, and entire sets. The predictive value of the EMT-related lncRNA signature was independent of age, The International Federation of Gynecology and Obstetrics stage, tumor grade, and body mass index. Time-dependent receiver operating characteristic curves also demonstrate the prognostic accuracy of this risk model. Gene Set Enrichment Analysis showed that cytokine-cytokine receptor interaction, glycolysis/gluconeogenesis, and IL-17 signaling pathway were significantly enriched. Furthermore, tumor microenvironment analysis indicated a significant negative correlation between the immune score and EMT-related lncRNA signature risks core, while the low-risk group was more likely to respond to ICB therapy than the high-risk group. A reliable EMT-related lncRNA signature of endometrioid EC was identified that could be utilized as an independent prognostic biomarker to predict patient survival outcomes and provide references for the option of ICB therapy.

High DNA methylation age deceleration defines an aggressive phenotype with immunoexclusion environments in endometrial carcinoma.

Like telomere shortening, global DNA hypomethylation occurs progressively with cellular divisions or in vivo aging and functions as a mitotic clock to restrain malignant transformation/progression. Several DNA-methylation (DNAm) age clocks have been established to precisely predict chronological age using normal tissues, but show DNAm age drift in tumors, which suggests disruption of this mitotic clock during carcinogenesis. Little is known about DNAm age alterations and biological/clinical implications in endometrial cancer (EC). Here we address these issues by analyzing TCGA and GSE67116 cohorts of ECs. Horvath clock analysis of these tumors unexpectedly revealed that almost 90% of them exhibited DNAm age deceleration (DNAmad) compared to patient chronological age. Combined with an additional clock named Phenoage, we identified a subset of tumors (82/429) with high DNAmad (hDNAmad+) as assessed by both clocks. Clinically, hDNAmad+ tumors were associated with advanced diseases and shorter patient survival, compared to hDNAmad- ones. Genetically, hDNAmad+ tumors were characterized by higher copy number alterations (CNAs) whereas lower tumor mutation burden. Functionally, hDNAmad+ tumors were enriched with cell cycle and DNA mismatch repair pathways. Increased PIK3CA alterations and downregulation of SCGB2A1, the inhibitor of PI3K kinase, in hDNAmad+ tumors, might promote tumor growth/proliferation and stemness. In addition, the inactivation of aging drivers/tumor suppressors (TP53, RB1, and CDKN2A) while enhanced telomere maintenance occurred more frequently in hDNAmad+ tumors, which supports sustained tumor growth. Prominently, hDNAmad+ tumors were featured with immunoexclusion microenvironments, accompanied by significantly higher levels of VTCN1 expression while lower PD-L1 and CTLA4 expression, which indicates their poor response to immune checkpoint inhibitor (ICI)-based immunotherapy. We further showed significantly higher levels of DNMT3A and 3B expression in hDNAmad+ than in hDNAmad- tumors. Thus, the tumor suppressive function of aging-like DNA hypomethylation is severely impaired in hDNAmad+ tumors, likely due to enhanced expression of DNMT3A/3B and dysregulated aging regulators. Our findings not only enrich biological knowledge of EC pathogenesis but also help improve EC risk stratification and precision ICI immunotherapy.

Novel insights into tumorigenesis and prognosis of endometrial cancer through systematic investigation and validation on mitophagy-related signature.

In-depth studies on the pathogenesis of endometrial cancer (EC) are critical because of the increasing global incidence of EC. Mitophagy, a mitochondrial quality control process, plays an important role in carcinogenesis and tumor progression. This study aimed to develop a novel mitophagy-based signature to predict the tumorigenesis and prognosis of EC. Data was downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases, and 29 mitophagy-related genes were downloaded from the Pathway Unification Database. EC patients were classified into two risk groups based on the two-key- gene signature, TOMM40 and MFN1, which were constructed using Cox regression analysis. A better prognosis was noted in the low-risk group. The model was validated for four aspects: clinical features, mutation status, clinical therapeutic response, and immune cell infiltration status. Moreover, according to the contribution to the risk model, TOMM40 was selected for further in vitro experiments. The silencing of TOMM40 inhibited mitochondrial degradation; suppressed cell proliferation; induced cell apoptosis and G1 phase cell cycle arrest; inhibited migration, invasion, and epithelial-mesenchymal transition; and suppressed cell stemness. In conclusion, the mitophagy-related risk score provides a novel perspective for survival and drug selection during the individual treatment of EC patients. TOMM40 serves as an oncogene in EC and promotes tumor progression via a mitophagy-related pathway. Thus, TOMM40 is a potential therapeutic target in EC.

A phase 2, two-stage study of avelumab and axitinib in patients with mismatch repair–proficient (MMR-P) recurrent or persistent endometrial cancer (EC).

TPS5628 Background: Despite significant strides in understanding the molecular pathogenesis of EC, there remain few effective therapies for recurrent disease. Deeper insight into the roles of disordered tumor vasculature and HIF1α- and VEGF-mediated immunosuppressive effects on myeloid-derived suppressor cells, T-cells, and PD-L1 expression contributed to the development of new targeted regimens. By inhibiting VEGF receptor (VEGFR) and PD-L1 signaling, immunologically “cold” tumors may become inflamed. The combination of the anti-PD-L1 antibody avelumab with axitinib, an inhibitor of VEGFR 1-3 and PDGFR with more potent IC50 inhibitory activity than lenvatinib, has also shown synergistic activity and is FDA approved as first line treatment for patients with renal cell cancer. We therefore hypothesized that this combination would be well tolerated and efficacious in recurrent MMR-P EC. Objectives:This is a non-randomized two-stage phase 2 study in patients with MMR-P EC evaluating the activity of combined avelumab and axitinib, as assessed by the co-primary endpoints of (1) frequency of patients surviving progression-free for at least 6 months (PFS6), and (2) the objective response rate (ORR) as measured by RECIST 1.1. Methods: Patients must have MMR-P EC and have received at least one chemotherapeutic regimen, with no upper limit on the number of prior lines received. Prior use of immune checkpoint inhibitors (ICI) is excluded. Eligible patients received avelumab 800 mg IV every 2 weeks and axitinib 5 mg orally twice daily until disease progression or unacceptable toxicity. In the first stage of the study, 16 patients were enrolled; if there were ≥2 objective responses or ≥2 PFS6 responses, accrual continued to the second stage with enrollment of an additional 19 patients. The combination of avelumab/axitinib would be considered worthy of further study if overall there were ≥4 objective responses or ≥8 PFS6 responses. Results: As of the data-cutoff in September 2022, a total of 28 patients had received study treatment; one patient withdrew from the study due to toxicity, prior to her first radiographic assessment. The median number of prior lines was 1 (range 1-4). Median follow-up was 12.7 months. Of the 28 patients receiving study treatment, eleven patients (39.3%, 95% CI 21.5%-59.4%) achieved a PR [11/28; 8 confirmed PR (28.6%) and 3 unconfirmed PR (10.7%)]. An additional 39.3% (11/28) had stable disease as best response. Median PFS was 7.3 months (95% CI 3.9-9.0). PFS6 was 59.3% (95% CI 35.7%-76.7%) and at 6 months, 10 patients were still alive and progression-free. Updated efficacy data and safety data will be reported at the time of the meeting. Conclusions: With 8 confirmed PRs and 10 PFS6 responses, the co-primary endpoints were met and the combination of avelumab/axitinib is considered worthy of further study in this population of MMR-P EC. Clinical trial information: NCT02912572 .

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

Abstract 4236: Obesity-associated endometrial cancer: Identifying the contributions of TMEM205 expression to the pathogenesis of disease

Abstract Background & Objective: Endometrial cancer (EC) is the most common gynecologic malignancy in the US. EC is more strongly associated with obesity than any other cancer type, with 57% of cases linked to obesity. As rates of obesity increase, the incidence of EC is also rising with worsening prognosis. The objective of this study is to identify the molecular mechanisms of TMEM205 expression that regulate exosome secretion, and the oncogenic proteins that underlie this transformation. Our findings are critical to understanding the pathways of obesity mediated EC and essential for developing novel strategies for preventing and treating EC. Methods: The exosomes were then quantified using nanoparticle tracking analyzer (NTA) and their size measured by Transmission electron microscopy (TEM). TMEM205, STAT5, FAS and PIAS3 expression was confirmed by IHC, WB and RT-PCR in patient and high fat diet treated mouse tissue. Endometrial hyperplasia was developed in immunocompetent mice using high fat diet (HFD; 45 kcal% fat diet) for 16 weeks. Results: To examine the effect of a continuous HFD (45 kcal% fat diet) on adipose, uterine and endometrial tissue, we first investigated body weight and uterine morphology in mice after 24 weeks of treatment. As expected, mice fed a HFD had a significantly higher body weight and increased adipose tissue when compared to control. In addition, mice in the HFD group showed marked enlargement in uterine horn size (hyperplasia) and inflammation with increased cell proliferation in the endometrial layer. The HFD treated mice were associated with increased exosome secretion and expression of the oncogenic proteins TMEM205 and STAT5. Expression of the tumor suppressor gene PIAS3 wasdownregulated in adipose and uterine tissues. L-2265 is a small molecule inhibitor that selectively inhibits TMEM205 and exosome secretion in EC cancer cells. To examine the effect of L-2263 in the context of obesity, we fed mice a HFD and treatment with L-2263 (2mg/kg/) weekly for 3 doses. L-2263 treatment resulted in significantly decreased body weight (BW) and decreased accumulation of adipose tissue relative to control HFD treated mice. Mice in the HFD group showed marked enlargement in uterine horn size (hyperplasia), whereas mice that received L-2263 treatment had normal uterine morphology of mouse age. L-2263 treated mice also demonstrated a significant decrease in the expression of proteins involved in exosome secretion (e.g., TMEM205 and STAT5) and those found in exosomes. Conclusion: This study provides fundamental insight into the mechanism of obesity-mediated TMEM205 expression and exosome secretion and their contribution to the pathogenesis of EC. This study is also provides pre-clinical evidence for the development of the first in-human studies of exosome-targeted therapies to prevent obesity-mediated EC. Citation Format: Takahiko Sakaue, Roman Zingarelli, Wafa Khadraoui, Uksha Saini, John Wallbillich, Muralidharan Anbazhakan, Ross Warner, Kalpana Deepa Priya Dorayappan, Deepika Kalaiyarasan, Molly Morton, Casey Cosgrove, Adrian Suarez, Larry J. Maxwell, David O'Malley, David E. Cohn, Karuppaiyah Selvendiran. Obesity-associated endometrial cancer: Identifying the contributions of TMEM205 expression to the pathogenesis of disease. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4236.

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.

Chasing Uterine Cancer with NK Cell-Based Immunotherapies

Gynecological cancers, including endometrial adenocarcinoma, significantly contribute to cancer incidence and mortality worldwide. The immune system plays a significant role in endometrial cancer pathogenesis. NK cells, a component of innate immunity, are among the critical innate immune cells in the uterus crucial in menstruation, embryonic development, and fighting infections. NK cell number and function influence endometrial cancer development and progression. Hence, it becomes crucial to understand the role of local (uterine) NK cells in uterine cancer. Uterine NK (uNK) cells behave differently than their peripheral counterparts; for example, uNK cells are more regulated by sex hormones than peripheral NK cells. A deeper understanding of NK cells in uterine cancer may facilitate the development of NK cell-targeted therapies. This review synthesizes current knowledge on the uterine immune microenvironment and NK cell-targeted uterine cancer therapeutics.

RNA m6A methylation regulators in endometrial cancer (Review).

As one of the three major malignant tumor types of the female reproductive system, endometrial cancer (EC) is the most prevalent gynecologic cancer in developed countries. In recent years, the incidence of EC has increased worldwide, threatening the health and well‑being of women. Recent research has indicated that the expression of multiple N6‑methyladenosine (m6A) regulators is up‑ or downregulated in EC and that abnormalities in m6A methylation and the expression of associated regulators are critical to the pathogenesis and progression of EC. m6A is the most abundant internal modification of mRNA. Several studies have demonstrated a close association between the development and progression of malignant tumors and the epigenetic phenomenon of m6A methylation. In the present study, the current status of research on m6A methylation in EC was reviewed. The mechanisms of methyltransferase, demethylase and m6A binding protein in regulating the development and progression of EC by modifying mRNA were introduced. The related research results will provide novel methods and approaches for the prevention and treatment of EC.

Beyond Serous: Treatment Options for Rare Endometrial Cancers.

Rare endometrial cancers are high-grade, aggressive malignancies which are often diagnosed at an advanced stage, and account for disproportionately more deaths than their more common low-grade counterparts. Standard of care includes a combination of surgery, radiation, and chemotherapy. Surgery consists of complete hysterectomy, and more recent evidence supports replacing a full lymphadenectomy with sentinel node mapping. Paclitaxel and carboplatin remain the mainstays of chemotherapy, while current studies incorporating immunotherapy will inform future practice. Whether and how to incorporate radiation remains controversial, and certain histologic subtypes, such as carcinosarcomas, may benefit from radiation more than others. Owing to their relative rarity, it is difficult to conduct clinical trials in this patient population, which has hindered the development of effective therapies for rare malignancies. Molecular profiling has offered insight into the pathogenesis of rare endometrial cancers, providing actionable targets for personalized therapy.