Endometrial Carcinoma Cells Research Articles

MGAT4A/Galectin9-Driven N-Glycosylation Aberration as a Promoting Mechanism for Poor Prognosis of Endometrial Cancer with TP53 Mutation.

Emerging evidence recognizes aberrant glycosylation as the malignant characteristics of cancer cells, but little is known about glycogenes' roles in endometrial carcinoma (EC), especially the most aggressive subtype carrying TP53 mutations. Using unsupervised hierarchical clustering, an 11-glycogene cluster is identified to distinguish an EC subtype associated with frequent TP53 mutation and worse prognosis. Among them, MGAT4A (alpha-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase A) emerges as the most consistently overexpressed glycogene, contributing to EC aggressiveness. In the presence of galectin-9, MGAT4A increases EC cell proliferation and invasion via promoting glucose metabolism. N-glycoproteomics further revealed GLUT1, a glucose transporter, as a glycoprotein modified by MGAT4A. Binding of galectin-9 to the MGAT4A-branched N-glycan on GLUT1 enhances its cell membrane distribution, leading to glucose uptake increase. In addition, oncogenic mutations of TP53 gene in EC cells upregulate MGAT4A expression by disrupting the regulatory oversight exerted by wild-type p53 on tumor-suppressive miRNAs, including miR-34a and miR-449a/b. The findings highlight a new molecular mechanism involving MGAT4A-regulated N-glycosylation on the key regulator of glucose metabolism in p53 mutants-driven EC aggressiveness, which may provide a strategic avenue to combat advanced EC.

Spatio-temporal localization of P21-activated kinase in endometrial cancer.

Endometrial cancer is the sixth most common gynecologic cancer, and has been reported as a malignancy arising due to the idiopathic effects of certain anticancer agents. Tamoxifen is the drug of choice in ER-positive breast cancer, and several studies have shown better disease-free survival in these patients. However, the long-term usage of tamoxifen has been associated with resistance and risk for endometrial malignancy. A direct mechanistic basis for tamoxifen-induced endometrial tumorigenesis is still unclear. Hyperactivation of PAK1 in endometrial cancer correlates with poor overall survival. The present study demonstrates that tamoxifen treatment induces nuclear localization of PAK1 in endometrial carcinoma cells. This nuclear transit is mediated through JAK2 phosphorylation of PAK1 and binding of β-PIX. In addition, a computational approach involving molecular modeling and simulation of phosphorylated and unphosphorylated forms of PAK1 was used to elucidate the dynamics of nuclear localization. Thus, PAK1 phosphorylation by JAK2 is a prerequisite for its nuclear localization and its tumorigenic effects on endometrial cancer cells.

Retracted] Stimulation of peroxisome proliferator‑activated receptor γ inhibits estrogen receptor α transcriptional activity in endometrial carcinoma cells.

Following the publication of this paper, after having performed an independent analysis of the data shown in the various of the figures in the Editorial Office, it was identified that, in Fig. 5A and B on p. 1232 showing the results of Transwell invasion and migration experiments, the majority of the data panels exhibited overlapping sections of data, such that the affected panels, which were intended to show the results of differently performed experiments, had all apparently been derived from the same original sources. Owing to the large number of duplications of data that have been identified in this paper, the Editor of Oncology Reports has decided that it should be retracted from the Journal on account of a lack of confidence in the presented data. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 33: 1227‑1234, 2015; DOI: 10.3892/or.2015.3729].

Molecular classification of clear cell endometrial carcinoma

Molecular classification of clear cell endometrial carcinoma

RNF144A as a potential risk gene for endometrial carcinoma: Insights from Mendelian randomization, bulk RNA sequencing, single-cell RNA, and experimental analysis.

Endometrial carcinoma (EC) is a prevalent gynecological malignancy that poses a significant threat to women's health worldwide. However, its pathogenesis and underlying mechanisms remains unclear. In this study, expression quantitative trait loci data, Mendelian randomization analysis, and differential expression analysis were performed to identify potential targets. A prognostic risk signature was subsequently constructed for EC patients based on the expression of these genes. Four bioinformatics algorithms, including generalized linear model, extreme gradient boosting, support vector machine, and random forest, were used to identify hub genes in EC. The expression of ring finger protein 144A (RNF144A) was validated using quantitative real-time polymerase chain reaction. Cellular proliferation and migration ability were evaluated using CCK-8 and Transwell assays, respectively. The genes RNF144A, ketohexokinase, and Rab interacting lysosomal protein like 2 were identified as potential targets for EC. Their differential expression was observed in EC patients, and Mendelian randomization analysis revealed a negative correlation between these genes and the development of EC. Mechanistic analyses suggested a strong association between these genes and the tumor immune microenvironment. The constructed risk signature was significantly associated with the prognosis, age, cancer stage, and grade of EC patients. Furthermore, based on interacted model algorithms, RNF144A was identified as a hub gene in EC. It was found to be significantly downregulated in EC samples, and its expression was positively correlated with the stage and grade of EC patients. In vitro experiments showed that overexpression of RNF144A significantly promoted cell growth and migration in EC cells. In conclusion, this study provides insights into the molecular mechanisms underlying EC progression and identifies preliminary candidate biomarkers for the development of EC treatment strategies.

Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

This study aimed to evaluate the expression and clinical significance of budding uninhibited by benzimidazole 1 (BUB1) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) in endometrial carcinoma (EC). BUB1 and BUBIB expressions were evaluated by bioinformatics. Protein expression, clinical features, prognosis and immune cell infiltration were explored in 20 EC tumors. siRNA was used to evaluate BUB1 and BUBIB function in EC cells. BUB1 and BUBIB were highly expressed in 26 cancers. BUB1 was associated with overall survival (OS) in eight cancers and disease-free survival in ten; BUB1B was associated with OS in nine cancers and DFS in eleven. BUB1 and BUBIB exhibited high frequencies of gene changes (mainly mutations, > 5%) in cancer. BUB1 was negatively correlated and BUB1B was positively correlated with cancer-associated fibroblasts and endothelial cell infiltration. BUB1 and BUBIB knockdown decreased migration and invasion in EC cells. High BUB1 expression correlated with tumor malignant phenotypes (P < 0.05). High BUB1 mRNA expression reduced OS (P = 0.00036) and recurrence-free survival (P = 0.0011). High BUB1B mRNA expression reduced OS (P = 0.0024). BUB1/BUB1B correlated with activated CD8 + T and CD4 + T cell infiltration. BUB1 and BUBIB are highly expressed and correlated with clinicopathological characteristics in EC. BUB1 and BUBIB are potential prognosis markers and immunotherapy targets.

Cancer Frequency in MuSK Myasthenia Gravis and Histological Evidence of Paraneoplastic Etiology.

Cancer frequency in muscle-specific kinase myasthenia gravis (MuSK-MG) has not yet been explored and the mechanisms leading to the formation of MuSK IgG remain elusive. We aimed to explore cancer frequency in MuSK-MG patients and to assess MuSK expression in cancer cells from 2 tumors occurred in this cohort. Immunohistochemistry on tumor specimens revealed the expression of MuSK in the cancer cells from primary mediastinal B cell lymphoma and endometrial carcinoma. Twenty-one males and 73 females were enrolled. Fifteen cancers occurred in 13 of 94 patients (13.8%). Patients with cancer were significantly older at time of MuSK-MG onset. ANN NEUROL 2024;96:1020-1025.

The Effects of Lithium, Metformin and Everolimus Substances on Cell Growth in 2D and 3D Ishikawa Endometrial Carcinoma Cell Culture

Purpose: Our aim is to study the effects of the single and combined treatments of Everolimus, Metformin, and Lithium Chloride in two-dimensional (2D, monolayer) and three-dimensional (3D, spheroid) cell cultures of Ishikawa cells, which comprise the endometrial cancer cell line. Materials and methods: As part of the study, the effects of single and combined forms of Everolimus, Metformin, and Lithium Chloride were determined on cell viability, invasion, colony formation and apoptosis, and PI3K/AKT/mTOR pathway. Cell viability was assessed using XTT assay. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genes were evaluated with RT-PCR, apoptosis was evaluated by flow cytometry and 3D spheroid results were evaluated with invert microscope analysis. Results: Everolimus, metformin, and lithium's IC50 levels were found at 48 hours to be 37.46 nM, 48.59 mM, and 100 µM, respectively. It was determined that the invasive capacities of Ishikawa cells in treatment groups, as well as cell colony formation were significantly reduced. In addition, Ishikawa spheroid cells were significantly suppressed compared with the control groups. RT-PCR results revealed that substances and their combinations affect genes associated with PI3K/AKT/mTOR pathway and apoptosis. Flow cytometry results showed notably increased apoptosis by single and combined treatments. Conclusion: As a result, the single and combination forms of everolimus, metformin, and lithium have reduced cell proliferation, induced apoptosis, and decreased mTOR activation through various mechanisms in Ishikawa cells.

CDK1 Acts as a Prognostic Biomarker Associated with Immune Infiltration in Pan-Cancer, Especially in Gastrointestinal Tumors.

Cyclin-dependent kinase 1 (CDK1) regulates the cell cycle and is highly expressed in most tumors. CDK1 expression has been associated with poor disease prognosis. This study aimed to identify the prognostic value of CDK1 in pan-cancer and investigate the association between CDK1 expression and immune cell infiltration. CDK1 expression and its correlation with prognosis in pan-cancer were analyzed using online databases. Immune infiltration was assessed by ESTIMATE and CIBERSORT algorithms. We then evaluated the relationship between CDK1 expression and tumor mutational burden (TMB), microsatellite instability (MSI), or tumor-infiltrating immune cells. In addition, we performed the co-expression analysis of immune-related genes and GO analysis with CDK1 expression in pan-cancer. Finally, we compared the CDK1 expression profile with the immune-related genes in 30 pairs of clinical gastrointestinal tumor samples. Our analysis demonstrated overexpression of CDK1 in most tumor tissues, especially in gastrointestinal tumors. The high expression of CDK1 was associated with poor overall survival, disease-specific survival, disease-free interval, and progression-free interval in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), and sarcoma (SARC). Besides, CDK1 expression was significantly associated with TMB in 22 cancer types and MSI in 8 cancer types as well as greater frequencies of MSI-high (MSI-H) status and high tumor mutational burden (TMB-H) in uterine corpus endometrial carcinoma (UCEC), stomach adenocarcinoma (STAD), sarcoma (SARC), rectum adenocarcinoma (READ), mesothelioma (MESO), head and neck squamous cell carcinoma (HNSC), and colon adenocarcinoma (COAD). In addition, CDK1 expression correlated with immune cell infiltrating levels, such as M0, M1, or M2 macrophages, memory CD4 T cells, T follicular helper cells, and naive B cells. Our data showed that CDK1 was remarkably correlated with 47 immune-related and immune checkpoint genes in many cancer types. Furthermore, CDK1 was up-regulated in gastrointestinal tumor samples, especially in gastric cancer and intestinal cancer. CDK1 was positively correlated with IDO1 in gastric cancer and PD-1 in intestinal cancer. Taken together, our data demonstrated the roles of CDK1 in oncogenesis and metastasis in pan-cancer. Thus, CDK1 is a potential prognostic biomarker and a target for tumor immunotherapy.

Upregulation of sperm-associated antigen 5 expression in endometrial carcinoma was associated with poor prognosis and immune dysregulation, and promoted cell migration and invasion

Sperm-associated antigen 5 (SPAG5) regulates cancer cell invasion and is involved in the progression of many cancers. However, the role of SPAG5 in endometrial carcinoma (EC) is still unknown. The purpose of this study was to explore the role of SPAG5 in EC and its potential molecular mechanism. The UALCAN tool and cBioPortal were used to analyze the expression and alterations of SPAG5 in EC, respectively. OncoLnc was used for survival analysis. We analyzed the effects of SPAG5 on immune cell infiltration and the expression levels of immune checkpoints. We also overexpressed and knocked down SPAG5 in EC cells to explore the effect of SPAG5 regulation on migration, invasion, apoptosis, and the cell cycle of EC cells. We found that SPAG5 was overexpressed and the SPAG5 gene was often mutated in EC. High SPAG5 expression was significantly associated with poor overall survival in patients with EC. SPAG5 also affected the level of immune cell infiltration in the TIME and the expression of immune checkpoints lymphocyte activating 3 (LAG3) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) in patients with EC. It may also be involved in the immunotherapy response in these patients. In vitro experiments showed that SPAG5 promotes cancer cell migration and invasion. In conclusion, this study lays the foundation for further understanding the molecular mechanisms of EC involving SPAG5 and contributes to diagnosing and managing this disease.

In silico design of novel multitarget small molecule inhibitors to treat PTEN-mutant endometrial carcinoma.

e15119 Background: Previously we described triple CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We used in silico modeling to optimize LCI133’s physicochemical properties to increase PI3K p110α potency/ADME to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety in normal cell lines. Methods: LCI139 + analogs were designed using computational chemistry based on X-ray crystal structures of each target protein + validated chemistry using nuclear magnetic resonance + high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay + selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours + stained with Annexin V/7-AAD to assess apoptosis. Representative PTEN wildtype (HEC-1A) + mutant (AN3CA) EC cell lines were chosen for mechanistic studies + treated 6 to 24 hours with multitarget or single agent inhibitor. Cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer. Cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da + is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 216.0nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 2.5nM) had nanomolar LCI139 IC50 values. PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis + colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP in PTEN mutant AN3CA. In PTEN-wildtype HEC1A WB, dose-dependent expression of pAKT in PTEN siRNA was noted. LCI139 generated heterogenous cell line specific effects on cell cycle progression + arrest. LCI139 displayed favorable toxicity against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines + displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 + flavopiridol. Our results merit mechanistic dissection of PTEN interaction with PI3K + CDK9 signaling pathways, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC.

Retrospective evaluation of drug to drug interactions with paclitaxel use in military health.

e24118 Background: Paclitaxel is an antineoplastic agent used in the management of multiple metastatic and non-resectable tumor types approved for ovarian, breast, AIDS-related Kaposi sarcoma, non-small cell lung, pancreatic, cervical, endometrial, and thymoma or thymic carcinoma cancer by the US Food and Drug Administration. Cytochrome P450 (CYP) 3A4 or 2C8 inducers, inhibitors, or substrates may alter paclitaxel metabolism. Because DDIs can have an effect on clinical outcomes, we aimed to identify drugs that interact with paclitaxel and describe their clinical manifestations. Methods: A retrospective analysis was performed on the health records of patients in the US Department of Defense Cancer Registry (retrieved March 2022), Comprehensive Ambulatory/Professional Encounter Records, and Pharmacy Data Transaction Service database (both retrieved October 2022). Patients’ medical history, diagnoses, and demographics were extracted and analyzed for differences in adverse effects when these agents were used alone vs concomitantly with other prescription drugs. Patients’ diagnoses and prescription drug use were extracted to compare completed vs discontinued treatment groups, identify medications commonly co-administered with paclitaxel, and evaluate DDIs with antidepressants. Results: Of 702 patients using paclitaxel, 338 completed treatments. The P value for paclitaxel discontinuation when used alone vs concomitantly or 1 drug (alone or before or after) vs combination (2 to 4 drugs) was &lt; .001. Patients who took paclitaxel concomitantly with a greater number of prescription drugs had a higher rate of treatment discontinuation than those who received fewer medications. Patients in the completed group received 9 to 56 prescription drugs, and those in the discontinued group were prescribed 6 to 70. Those who discontinued treatment had more diagnosed medical issues than those who completed treatment. Conclusions: This review cannot conclude that concomitant use with prescription drug(s) resulted in paclitaxel discontinuation. There were no significant DDIs determined between paclitaxel and antidepressants.[Table: see text]

NLRC5 promotes endometrial carcinoma progression by regulating NF-κB pathway-mediated mismatch repair gene deficiency

The innate immune molecule NLR family CARD domain-containing 5 (NLRC5) plays a significant role in endometrial carcinoma (EC) immunosurveillance. However, NLRC5 also plays a protumor role in EC cells. Mismatch repair gene deficiency (dMMR) can enable tumors to grow faster and also can exhibit high sensitivity to immune checkpoint inhibitors. In this study, we attempted to determine whether NLRC5-mediated protumor role in EC is via the regulation of dMMR. Our findings revealed that NLRC5 promoted the proliferation, migration, and invasion abilities of EC cells and induced the dMMR status of EC in vivo and in vitro. Furthermore, the mechanism underlying NLRC5 regulated dMMR was also verified. We first found NLRC5 could suppress nuclear factor-kappaB (NF-κB) pathway in EC cells. Then we validated that the positive effect of NLRC5 in dMMR was restricted when NF-κB was activated by lipopolysaccharides in NLRC5-overexpression EC cell lines. In conclusion, our present study confirmed the novel NLRC5/NF-κB/MMR regulatory mechanism of the protumor effect of NLRC5 on EC cells, thereby suggesting that the NLRC5-mediated protumor in EC was depend on the function of MMR.

Long non-coding RNA JPX promotes endometrial carcinoma progression via janus kinase 2/signal transducer and activator of transcription 3.

Although LncRNA JPX has been linked to a number of malignancies, it is yet unknown how it relates to endometrial carcinoma (EC). Investigating the expression, functional activities, and underlying molecular processes of lncRNA JPX in EC was the goal of this work. RT-qPCR was used to examine the differences in lncRNA/microRNA (miRNA, miR)/mRNA expression between normal cervical and EC tissues or cells. Cell Counting Kit-8, flow cytometry, and transwell were used to evaluate the association between lncRNA JPX/miR-140-3p/phosphoinositide-3-kinase catalytic subunit α (PIK3CA) in Ishikawa and JEC cell lines. The impact of JPX on the downstream janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 signaling pathway was investigated using Western blot analysis. When comparing EC tissues to nearby normal tissues, JPX expression is markedly increased in EC tissues, with greater expression in advanced-stage EC. Furthermore, compared to normal epithelial cells, EC cell lines have higher levels of JPX expression. In Ishikawa and JEC endometrial cancer cell lines, we used siRNA-mediated suppression of JPX to find lower cell viability, increased apoptosis, cell cycle arrest, and reduced migration and invasion. We next verified that miR-140-3p binds to downstream target cells to impede the transcription and translation of PIK3CA, which in turn prevents the growth of Ishikawa and JEC cells. JPX functions as a ceRNA to adsorb miR-140-3p. This procedure required controlling JAK2/STAT3, a downstream signal. JPX enhances the development of Ishikawa and JEC cells and activates downstream JAK2/STAT3 signal transduction via the miR-140-3p/PIK3CA axis, offering a possible therapeutic target for the treatment of EC.

The prognostic value of lymph node to primary tumor standardized uptake value ratio in cancer patients: a meta-analysis.

The lymph node to primary tumor standardized uptake value ratio (NTR) is an innovative parameter derived from positron emission tomography/computed tomography (PET/CT) scans that captures the intricate relationship between primary tumors and associated lymph nodes. This meta-analysis aimed to investigate the prognostic value of NTR in cancer patients. A systematic search of PubMed, Cochrane, and Embase databases was conducted to identify studies investigating the association between NTR and survival outcomes in cancer patients. The pooled adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) were calculated using a random-effects model. Twelve studies comprising a total of 2037 patients were included in the meta-analysis. Elevated NTR was significantly associated with worse overall survival aHR (2.21, 95% CI 1.63 to 2.99), disease-free survival aHR (3.27, 95% CI 2.12 to 5.05), and distant metastasis-free survival aHR (2.07, 95% CI 1.55 to 2.78) in cancer patients. Subgroup analyses by cancer type showed consistent results across various malignancies, including head and neck squamous cell carcinoma, endometrial carcinoma, lung cancer, breast cancer, and nasopharyngeal carcinoma. This meta-analysis provides evidence for a significant association between elevated NTR and worse survival outcomes in cancer patients. Elevated NTR may serve as a useful prognostic biomarker for cancer patients and could potentially be used to guide treatment decisions and monitor disease progression. Future studies should aim to validate these findings in larger and more diverse patient populations and investigate the underlying mechanisms for the observed association between NTR and survival outcomes.

Glycometabolism and lipid metabolism related genes predict the prognosis of endometrial carcinoma and their effects on tumor cells

BackgroundGlycometabolism and lipid metabolism are critical in cancer metabolic reprogramming. The primary aim of this study was to develop a prognostic model incorporating glycometabolism and lipid metabolism-related genes (GLRGs) for accurate prognosis assessment in patients with endometrial carcinoma (EC).MethodsData on gene expression and clinical details were obtained from publicly accessible databases. GLRGs were obtained from the Genecards database. Through nonnegative matrix factorization (NMF) clustering, molecular groupings with various GLRG expression patterns were identified. LASSO Cox regression analysis was employed to create a prognostic model. Use rich algorithms such as GSEA, GSVA, xCELL ssGSEA, EPIC,CIBERSORT, MCPcounter, ESTIMATE, TIMER, TIDE, and Oncoppredict to analyze functional pathway characteristics of the forecast signal, immune status, anti-tumor therapy, etc. The expression was assessed using Western blot and quantitative real-time PCR techniques. A total of 113 algorithm combinations were combined to screen out the most significant GLRGs in the signature for in vitro experimental verification, such as colony formation, EdU cell proliferation, wound healing, apoptosis, and Transwell assays.ResultsA total of 714 GLRGs were found, and 227 of them were identified as prognostic-related genes. And ten GLRGs (AUP1, ESR1, ERLIN2, ASS1, OGDH, BCKDHB, SLC16A1, HK2, LPCAT1 and PGR-AS1) were identified to construct the prognostic model of patients with EC. Based on GLRGs, the risk model’s prognosis and independent prognostic value were established. The signature of GLRGs exhibited a robust correlation with the infiltration of immune cells and the sensitivity to drugs. In cytological experiments, we selected HK2 as candidate gene to verify its value in the occurrence and development of EC. Western blot and qRT-PCR revealed that HK2 was substantially expressed in EC cells. According to in vitro experiments, HK2 knockdown can increase EC cell apoptosis while suppressing EC cell migration, invasion, and proliferation.ConclusionThe GLRGs signature constructed in this study demonstrated significant prognostic value for patients with endometrial carcinoma, thereby providing valuable guidance for treatment decisions.

Abstract 1816: In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma

Abstract Background: Previously we described multitarget CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We have optimized LCI133’s chemical properties to increase PI3K p110α potency to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety against normal cell lines. Methods: LCI139’s chemical structure was determined using nuclear magnetic resonance and high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours and stained with Annexin V/7-AAD to assess apoptosis by cytometer. Representative PTEN wildtype (HEC-1) and mutant (AN3CA) EC cell lines were chosen for mechanistic studies and treated 6 to 24 hours with multitarget or single agent inhibitor. RNA was isolated to determine gene expression by qRT-PCR, cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer, or cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da and is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 243.6nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 9.8nM) had nanomolar LCI139 IC50 values, while PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis and colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP. MCL-1 ablation correlated to decreased Mcl1 expression (p&amp;lt;0.0010) assessed 6 hours post treatment. LCI139 generated heterogenous cell line specific effects on cell cycle progression and arrest. LCI139 displayed a favorable toxicity profile against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines and displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 and flavopiridol. Our results merit further mechanistic dissection of PTEN interaction with PI3K pathway signaling, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC. Citation Format: Vaidehi Mujumdar, Ritchie Delara, Hailey Dryden, Dhananjaya Pal, Krishnaiah Maddeboina, Page Mangum Arditti, Bharath Yada, Robert Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Donald Durden, Cody McHale. In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1816.

Abstract 2838: ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

As a dedifferentiated tumor, small cell endometrial neuroendocrine tumors (NETs) are rare and frequently diagnosed at an advanced stage with a poor prognosis. Current treatment recommendations are often extrapolated from histologically similar tumors in other sites or based on retrospective studies. The exploration for diagnostic and therapeutic markers in small cell NETs is of great significance. In this study, we conducted single-cell RNA sequencing on a specimen obtained from a patient diagnosed with small cell endometrial neuroendocrine carcinoma (SCNEC) based on pathology. We revealed the cell map and intratumoral heterogeneity of the cancer cells through data analysis. Further, we validated the function of ISL LIM Homeobox 1 (ISL1) in vitro in an established neuroendocrine cell line. Finally, we examined the association between ISL1 and tumor staging in small cell lung cancer (SCLC) patient samples. We observed the significant upregulation of ISL1 expression in tumor cells that showed high expression of the neuroepithelial markers. Additionally, in vitro cell function experiments demonstrated that the high ISL1 expression group exhibited markedly higher cell proliferation and migration abilities compared to the low expression group. Finally, we showed that the expression level of ISL1 was correlated with SCLC stages. ISL1 protein in NETs shows promise as a potential biomarker for diagnosis and treatment.

Progesterone inhibits endometrial cancer growth by inhibiting glutamine metabolism through ASCT2.

Endometrial carcinoma (EC) is a common malignancy that originates from the endometrium and grows in the female reproductive system. Surgeries, as current treatments for cancer, however, cannot meet the fertility needs of young women patients. Thus, progesterone (P4) therapy is indispensable due to its effective temporary preservation of female fertility. Many cancer cells are often accompanied by changes in metabolic phenotypes, and abnormally dependent on the amino acid glutamine. However, whether P4 exerts an effect on EC via glutamine metabolism is unknown. In the present study, we found that P4 could inhibit glutamine metabolism in EC cells and down-regulate the expression of the glutamine transporter ASCT2. This regulation of ASCT2 affects the uptake of glutamine. Furthermore, the in vivo xenograft studies showed that P4 inhibited tumor growth and the expression of key enzymes involved in glutamine metabolism. Our study demonstrated that the direct regulation of glutamine metabolism by P4 and its anticancer effect was mediated through the inhibition of ASCT2. These results provide a mechanism underlying the effects of P4 therapy on EC from the perspective of glutamine metabolism.