Aldo-keto Reductase Family 1 Member C3 Research Articles

Synthesis and anti-inflammatory activity of benzimidazole derivatives; an in vitro, in vivo and in silico approach

Many non-steroidal anti-inflammatory drugs (NSAIDs) concurrently inhibit both COX-1 and COX-2, with a preference for specifically targeting COX-2 due to its significant involvement in various pathologies. In addition to COX enzymes, several other targets, including Aldose reductase, Aldo-ketoreductase family 1-member C2, and Phospholipase A2, have been identified as contributors to inflammation and a myriad of other diseases.In this context, a series of 2-substituted benzimidazole derivatives was synthesized and assessed for their anti-inflammatory potential through both in vitro and in vivo assays. Molecular docking studies were conducted to elucidate the mechanism of action of these compounds against COX enzymes and other therapeutic targets associated with NSAIDs, such as Aldose reductase, AIKRC, and Phospholipase A2.Among the synthesized compounds, B2, B4, B7, and B8 demonstrated IC50 values lower than the standard ibuprofen, as determined by the Luminol-enhanced chemiluminescence assay. Validation of these findings was achieved through an in vivo carrageenan-induced mice paw edema model, confirming a comparable anti-inflammatory effect to diclofenac sodium observed in vitro. Notably, these compounds exhibited significant binding affinity with all therapeutic targets investigated in this study.These results suggest that the newly synthesized derivatives possess noteworthy anti-inflammatory potential, warranting further exploration for the development of novel multi-targeting inhibitors.

Abstract 5958: Exploring AKR1C3 as a therapeutic target to overcome erlotinib resistance in lung adenocarcinoma

Abstract Introduction: Erlotinib has demonstrated promising efficacy in lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor mutation. However, the development of resistance to erlotinib poses a significant challenge in achieving long-lasting treatment responses. Aldo-keto reductase family 1 member C3 (AKR1C3) is associated with tumor progression, drug resistance, and unfavorable prognosis. This study aims to assess the potential of AKR1C3 as a therapeutic target for overcoming erlotinib resistance in LUAD. Methods: AKR1C3 expression was assessed in paired tumor tissues from 100 LUAD patients using reverse transcription quantitative polymerase chain reaction. Both in vitro and in vivo experiments were categorized into four treatment groups: a saline control (CTR), erlotinib (ER), an AKR1C3 inhibitor 3-(4-trifluoromethyl)phenylamino)benzoic acid (BA), and the combination of erlotinib with the AKR1C3 inhibitor (COM). An erlotinib-resistant cell line was established from the LUAD cell line HCC4006. The MTS assay was used to evaluate cell proliferation and viability. The combined effect of erlotinib and the AKR1C3 inhibitor was assessed using Synergyfinder and CompuSyn calculation software. Both cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of erlotinib resistance to LUAD were established in NSG mice. The mice received daily oral gavage administration of the drugs for 31 days, while tumor volume was monitored biweekly. Results: AKR1C3 expression was significantly upregulated by more than three-fold in LUAD tumor samples compared to normal counterparts (p < 0.0001). After 24 hours of treatment in erlotinib-resistant cells, the ER and BA groups exhibited inhibition rates of 7% and 9%, respectively, while the COM group demonstrated a substantially higher inhibition rate of 45%. Synergyfinder and CompuSyn analysis revealed a synergy score of 12 and a combination index of 0.5 for the COM group, indicating a robust synergistic effect between the two drugs. In the CDX model, the COM group exhibited a tumor volume reduction of 25% in erlotinib-resistant cells and 64% in parental cells compared to the CTR group. In the PDX model, the COM group displayed a significant reduction of 50.4% in tumor volume compared to the CTR group. There was no significant difference in the weight of body and internal organs among the four treatment groups in both xenograft models. Conclusions: AKR1C3 overexpression was observed in the tumor tissues of LUAD patients. The combination of erlotinib and AKR1C3 inhibitor showed promising efficacy in overcoming erlotinib resistance in both in vitro and in vivo experiments. These findings highlight AKR1C3 as a potential therapeutic target for managing erlotinib resistance in LUAD patients. Targeting AKR1C3 represents a novel approach to overcome resistance and enhance treatment outcomes in LUAD patients undergoing erlotinib therapy. Citation Format: William C. Cho, Kwan P. Li, Chi F. Wong, King Y. Fung, James C. Chow, Ka M. Cheung, Jeffrey C. Chan, Eunice Y. Lau. Exploring AKR1C3 as a therapeutic target to overcome erlotinib resistance in lung adenocarcinoma [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 5958.

Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys.

AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5mg/m2 in the first-in-human dose escalation study.

AKR1C3 in carcinomas: from multifaceted roles to therapeutic strategies.

Aldo-Keto Reductase Family 1 Member C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5) or prostaglandin F (PGF) synthase, functions as a pivotal enzyme in androgen biosynthesis. It catalyzes the conversion of weak androgens, estrone (a weak estrogen), and PGD2 into potent androgens (testosterone and 5α-dihydrotestosterone), 17β-estradiol (a potent estrogen), and 11β-PGF2α, respectively. Elevated levels of AKR1C3 activate androgen receptor (AR) signaling pathway, contributing to tumor recurrence and imparting resistance to cancer therapies. The overexpression of AKR1C3 serves as an oncogenic factor, promoting carcinoma cell proliferation, invasion, and metastasis, and is correlated with unfavorable prognosis and overall survival in carcinoma patients. Inhibiting AKR1C3 has demonstrated potent efficacy in suppressing tumor progression and overcoming treatment resistance. As a result, the development and design of AKR1C3 inhibitors have garnered increasing interest among researchers, with significant progress witnessed in recent years. Novel AKR1C3 inhibitors, including natural products and analogues of existing drugs designed based on their structures and frameworks, continue to be discovered and developed in laboratories worldwide. The AKR1C3 enzyme has emerged as a key player in carcinoma progression and therapeutic resistance, posing challenges in cancer treatment. This review aims to provide a comprehensive analysis of AKR1C3's role in carcinoma development, its implications in therapeutic resistance, and recent advancements in the development of AKR1C3 inhibitors for tumor therapies.

AKR1C3 silencing inhibits autophagy-dependent glycolysis in thyroid cancer cells by inactivating ERK signaling.

Thyroid cancer is a highly differentiated and poorly malignant tumor. Interfering with glycolysis has become an effective means of controlling cancer progression and autophagy is negatively correlated with glycolysis. Aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated to be highly expressed in thyroid cancer tissue and the higher AKR1C3 expression predicted the worse prognosis. We aimed to explore whether AKR1C3 could affect thyroid cancer progression by regulating autophagy-dependent glycolysis. AKR1C3 expression in thyroid cancer cells was detected by western blot. Then, AKR1C3 was knocked down by transfection with short hairpin RNA specific to AKR1C3 in the absence or presence of 3-methyladenine (3-MA) or PMA treatment. Cell cycle and apoptosis was detected by flow cytometry. Immunofluorescence staining was used to analyze LC3B expression. Extracellular acidification, glucose uptake and lactic acid secretion were detected. To evaluate the tumorigenicity of AKR1C3 insufficiency on thyroid cancer in vivo, TPC-1 cells with AKR1C3 knockdown were injected subcutaneously into nude mice. Then, cyclinD1 and Ki67 expression in tumorous tissues was measured by immunohistochemical analysis. Apoptosis was assessed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Additionally, the expression of proteins related to cell cycle, apoptosis, glycolysis, autophagy, and extracellular signal-regulated kinase (ERK) signaling in cells and tumor tissues was assessed by western blot. Highly expressed AKR1C3 was observed in thyroid cancer cells. AKR1C3 knockdown induced cell cycle arrest and apoptosis of TPC-1 cells. Besides, autophagy was activated and glycolysis was inhibited following AKR1C3 silencing, and 3-MA treatment restored the impacts of AKR1C3 silencing on glycolysis. The further experiments revealed that AKR1C3 insufficiency inhibited ERK signaling and PMA application reversed AKR1C3 silencing-induced autophagy in TPC-1 cells. The in vivo results suggested that AKR1C3 knockdown inhibited the development of subcutaneous TPC-1 tumors in nude mice and inactivated the ERK signaling. Collectively, AKR1C3 silencing inhibited autophagy-dependent glycolysis in thyroid cancer by inactivating ERK signaling.

Computational Insight and Anticancer Effect of Cinnamic Acid-Derivative Amide Compounds

In this research, a meticulous screening process was conducted using four servers: Drug Target Explorer, Swiss Target Predictor, SEA Predictor, and Target Hunter. The primary objective was to identify a series of potential biological targets related to the regulation of cell growth and apoptosis in cancer cells using cinnamic acid derivatives. This study focused on five specific targets, matrix metallopeptidase 9 (MMP9), apoptosis inducing factor (AIF), aldo-keto reductase family 1 member C3 (AKR1C3), aldo-keto reductase family 1 member B10 (AKR1B10), mitogenactivated protein kinase 14 (MAPK14), all of which are well known to play a significant role in cancer cell dynamics. To explore both molecular recognition and molecular dynamics, a series of in silico investigations (docking and molecular dynamics) were carried out using a collection of 14 cinnamic acid derivatives, including cinnamic acid phenethyl ester (CAPE) as a notable reference molecule due to its widely recognized anticancer effects. Furthermore, preliminary in vitro data revealed a potentially promising cytotoxic effect of (E)-N-[(3,4-dichlorophenyl)methyl]- 3-(4-phenoxyphenyl)-2-propenamide (LQM755) on a human gastric adenocarcinoma cell-line (AGS cells), which are characterized by the overexpression of the MMP9 protein. Therefore, the chemical compound LQM755 provides an initial perspective in the field of cancer therapy.

Zika virus remodelled ER membranes contain proviral factors involved in redox and methylation pathways

Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes. Here, we isolate ER membranes from ZIKV-infected cells and determine their proteome. Forty-six host cell factors are enriched in ZIKV remodeled membranes, several of these having a role in redox and methylation pathways. Four proteins are characterized in detail: thioredoxin reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and increasing stability of viral transmembrane proteins; adenosylhomocysteinase (AHCY) indirectly promoting m6A methylation of ZIKV RNA by decreasing the level of S- adenosyl homocysteine and thus, immune evasion. These results highlight the involvement of redox and methylation enzymes in the ZIKV life cycle and their accumulation at virally remodeled ER membranes.

The AKR1C3-Activated Prodrug, Achm-025, Eradicates Disease in Preclinical Models of Aggressive T-Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is exceptionally difficult to cure after relapse. T-ALL expresses significantly higher levels of the enzyme aldo-keto reductase family 1 member C3 (AKR1C3) compared with B-cell ALL. To exploit this finding, we developed a novel prodrug, ACHM-025, which is selectively activated by AKR1C3 to form a potent cell-entrapped DNA alkylating agent. ACHM-025 was designed to improve drug specificity and minimize toxicity observed with currently used DNA alkylating agents, such as cyclophosphamide (CPM), a prodrug which is activated systemically via liver enzymes. We evaluated the in vivo efficacy and AKR1C3 selectivity of ACHM-025 against a panel of 25 pediatric T-ALL patient-derived xenografts (PDXs) alongside standard-of-care therapy. Methods: AKR1C3 expression in T-ALL PDXs was determined by RNA-seq, immunoblotting and intracellular flow cytometry. For in vivo efficacy studies, PDXs were established as orthotopic models in immune-deficient NSG mice. Engraftment was assessed by enumerating the proportion of human versus mouse CD45 + cells (%huCD45 +) in the peripheral blood. Treatment commenced when the %huCD45 + reached ≥1% (Day 0), and events were defined as %huCD45 + ≥25% or leukemia-related morbidity. Drug efficacy was assessed by mouse event-free survival (EFS) and stringent objective response measures. For the single agent study, ACHM-025 (IP weekly, Days 0, 7, 14) was assessed against 25 T-ALL PDXs using a single mouse trial (SMT) format (one vehicle treated mouse, one drug treated mouse). For the consolidation therapy comparison, ACHM-025 (IP Days 0, 7) or CPM (IP Days 0, 7) combined with cytarabine (Ara-C; IP Days 0-4, 7-11) and 6-mercaptopurine (6MP; IP Days 0-4, 7-11) were assessed against a T-ALL PDX derived from a patient at relapse. For the relapsed/refractory (R/R) therapy study, ACHM-025 (IP Days 0, 7, 14) and nelarabine (IP Days 0-4, 14-18) were assessed against a T-ALL PDX derived from a patient at relapse. Results: ACHM-025 dose-limiting toxicity in cynomolgus monkeys was neutropenia, where the pharmacokinetic equivalent dose in NSG mice was well tolerated. We first evaluated the in vivo efficacy of ACHM-025 as a single agent using the SMT format across an extended panel of 25 T-ALL PDXs, to address the impact of genetic heterogeneity in pediatric ALL on drug response. Remarkably, 7/25 PDXs treated with ACHM-025 did not relapse over 250 days after the last treatment and a total of 22/25 T-ALL PDXs scored an objective response. In comparison, vehicle treated EFS ranged from 3-26 days for the 25 T-ALL PDXs. Importantly, AKR1C3 expression (mRNA, protein or intracellular) provides a predictive biomarker of efficacy, as ACHM-025 was significantly more effective against T-ALL PDXs with high AKR1C3 expression versus those with low AKR1C3 expression (p<0.0001). CPM is included in standard-of-care consolidation therapy for ALL in combination with Ara-C and 6MP. Comparing single agents, ACHM-025 was significantly more effective than CPM (T-C 25 vs. 57 days, p=0.0005, Table 1). The combination of ACHM-025, Ara-C and 6MP was significantly more effective than standard-of-care consolidation therapy (CPM, Ara-C and 6MP), more than doubling survival (T-C 36 vs. 75 days, p=0.0005, Table 1). Finally, we compared ACHM-025 with nelarabine, which is the only FDA approved agent for R/R T-ALL. Comparing single agents, ACHM-025 was substantially more effective than nelarabine (T-C 19 vs. 198 days, p=0.0005, Table 1). Importantly, relapsed disease remained sensitive to ACHM-025 re-treatment in vivo, with no evidence of acquired resistance. Remarkably, no mice treated with the ACHM-025/nelarabine combination relapsed over 250 days after the last treatment (Table 1). Conclusions: ACHM-025 exerted profound in vivo efficacy against T-ALL PDXs and eradicated the disease in 7 aggressive T-ALL PDXs. ACHM-025 was significantly more effective than CPM both as a single agent and when used in combination with Ara-C/6MP. Notably, ACHM-025 in combination with nelarabine was curative when used to treat a chemoresistant T-ALL PDX in vivo. The in vivo efficacy of ACHM-025 directly correlated with AKR1C3 expression levels, providing a predictive biomarker for response. These data provide strong preclinical evidence highlighting the potential of ACHM-025 as a targeted and effective therapy for aggressive forms of T-ALL.

Keratinocytes Exposed to Blue or Red Light: Proteomic Characterization Showed Cytoplasmic Thioredoxin Reductase 1 and Aldo-Keto Reductase Family 1 Member C3 Triggered Expression.

Several cell-signaling mechanisms are activated by visible light radiation in human keratinocytes, but the key regulatory proteins involved in this specific cellular response have not yet been identified. Human keratinocytes (HaCaT cells) were exposed to blue or red light at low or high irradiance for 3 days in cycles of 12 h of light and 12 h of dark. The cell viability, apoptotic rate and cell cycle progression were analyzed in all experimental conditions. The proteomic profile, oxidative stress and mitochondrial morphology were additionally evaluated in the HaCaT cells following exposure to high-irradiance blue or red light. Low-irradiance blue or red light exposure did not show an alteration in the cell viability, cell death or cell cycle progression. High-irradiance blue or red light reduced the cell viability, induced cell death and cell cycle G2/M arrest, increased the reactive oxygen species (ROS) and altered the mitochondrial density and morphology. The proteomic profile revealed a pivotal role of Cytoplasmic thioredoxin reductase 1 (TXNRD1) and Aldo-keto reductase family 1 member C3 (AKR1C3) in the response of the HaCaT cells to high-irradiance blue or red light exposure. Blue or red light exposure affected the viability of keratinocytes, activating a specific oxidative stress response and inducing mitochondrial dysfunction. Our results can help to address the targets for the therapeutic use of light and to develop adequate preventive strategies for skin damage. This in vitro study supports further in vivo investigations of the biological effects of light on human keratinocytes.

Screening and identification of genes related to ferroptosis in keratoconus

Corneal keratoconus (KC) is a dilated (ectatic) corneal disease characterized by a central thinning of the cornea, which causes protrusion into a conical shape that seriously affects vision. However, due to the complex etiology of keratoconus, its entire mechanism remains unclear and there is no mechanism-directed treatment method. Ferroptosis is a novel programmed cell death mechanism related to lipid peroxidation, stress, and amino acid metabolism, which plays a crucial role in various diseases. This study aimed to explore the relationship between keratoconus and ferroptosis, to provide new insights into the mechanism of keratoconus development, and potential treatment options based on further elucidation of this mechanism. The corresponding mRNA microarray expression matrix data of KC patients were obtained from GEO database (GSE204791). Weighted co-expression network analysis (WGCNA) and support vector machine recursive feature elimination (SVM-RFE) were selected to screen hub genes, which were overlapped with ferroptosis genes (FRGs) from FerrDb. GO and GSEA were performed to analyze differential pathways, ssGSEA was used to determine immune status, and then, feasible drugs were predicted by gene-drug network. Additionally, we predicted the miRNA and IncRNA of hub genes to identify the underlying mechanism of disease so as to predict treatment for the disease. The epithelial transcriptome from keratoconus tissue mRNA microarray data (GSE204791) was extracted for the main analysis, including eight epithelial cells and eight epithelial control cells. The differential genes that were overlapped by WGCAN, SVM-RFE and FRGs were mainly related to oxidative stress, immune regulation, cellular inflammation, and metal ion transport. Through further analysis, aldo–keto reductase family 1 member C3 (AKR1C3) was selected, and negatively correlated with mature CD56 natural killer (NK) cells and macrophages. Then, gene-drug interaction network analysis and miRNA prediction were performed through the website. It was concluded that four immune-related drugs (INDOMETHACIN, DAUNORUBICIN, DOXORUBICIN, DOCETAXEL) and a miRNA (has-miR-184) were screened to predict potential drugs and targets for disease treatment. To our knowledge, this was the first report of KC being associated with ferroptosis and prompted search for differential genes to predict drug targets of gene immunotherapy. Our findings provided insight and a solid basis for the analysis and treatment of KC.

Crystal structure determination, molecular docking, and molecular dynamics of arylal dimedones as potential inhibitors for castrate-resistant prostate cancer.

Increased androgen receptor (AR) signaling brought on by higher intratumoral androgen production and AR amplification is associated with castrate-resistant prostate cancer (CRPC). Cell proliferation in this case continues even during low expression of testosterone in the body. Aldo-keto reductase family 1 member C3 (AKR1C3) is one of the most elevated genes in CRPC and catalyzes the formation of powerful AR ligands from inactive forms. The current work aimed to use the x-ray method to investigate the ligand's crystal structure while also conducting molecular docking and molecular dynamics tests on the synthesized molecules against AKR1C3. As per the results obtained, the MM-PBSA binding energies of inhibitors 2,2'-((4-methoxyphenyl)methylene)bis(3,4-hydroxy-5,5-dimethylcyclohex-2-en-1-one is -132.456kJmol-1 and 2,2'-(phenylmethylene)bis(3-hydroxy-5,5-dimethylcyclohex-2-en-1-one is -81.017kJmol-1 . These results create a promising approach to drug design based on its fit to the structures of the receptor site rather than basing it on analogies to other active structures.

Single-cell RNA sequencing depicts metabolic changes in children with aplastic anemia.

Aplastic anemia (AA) is a bone marrow hematopoietic failure syndrome mediated by immune cells. The mechanism of this immune disorder is not well understood and therapeutic strategies still need to be improved. Studies have found that abnormalities in metabolisms promote the survival of AA cells. In recent years, an increasing number of studies have reported the immunosuppressive therapy for the treatment of AA. In this study, we analyzed the transcriptome of AA from peripheral blood compared with healthy donors by single-cell sequencing and identified the affected metabolic pathways including lysine degradation. We demonstrated that the metabolic abnormalities of T lymphocytes mainly focus on glycolysis/gluconeogenesis. In addition, the metabolic abnormalities of natural killer cells concentrated in oxidative phosphorylation. The key genes involved in abnormal metabolic processes were Neustein neurotrophic factor (NENF), inositol polyphosphate-4-phosphatase type II B (INPP4B), aldo-keto reductase family 1, member C3 (AKR1C3), and carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 (CHST2) by differential gene expression analysis. Molecule interaction analysis showed that tumor necrosis factor superfamily, member 12 (TNFSM12) in tumor necrosis factor (TNF) signaling was broadly activated in AA. In conclusion, we suppose that the treatment of the immune cells' abnormal metabolic pathway may contribute to the development of novel strategies to treat AA.

Bioinformatics analysis of ferroptosis-related gene AKR1C3 as a potential biomarker of asthma and its identification in BEAS-2B cells

Ferroptosis is a newly discovered type of cell death and has recently been shown to be associated with asthma. However, the relationship between them at the genetic level has not been elucidated via informatics analysis. In this study, bioinformatics analyses are conducted using asthma and ferroptosis datasets to identify candidate ferroptosis-related genes using the R software. Weighted gene co-expression network analysis is performed to identify co-expressed genes. Protein–protein interaction networks, the Kyoto encyclopedia of genes and genomes, and gene ontology enrichment analysis are used to identify the potential functions of the candidate genes. We experimentally validate the results of our analysis using small interfering RNAs and plasmids to silence and upregulate the expression of the candidate gene in human bronchial epithelial cells (BEAS-2B). The ferroptosis signature levels are examined. Bioinformatics analysis of the asthma dataset GDS4896 shows that the level of the aldo-keto reductase family 1 member C3 (AKR1C3) gene in the peripheral blood of patients with severe therapy-resistant asthma and controlled persistent mild asthma (MA) is significantly upregulated. The AUC values for asthma diagnosis and MA are 0.823 and 0.915, respectively. The diagnostic value of AKR1C3 is verified using the GSE64913 dataset. The gene module of AKR1C3 is evident in MA and functions through redox reactions and metabolic processes. Ferroptosis indicators are downregulated by the overexpression of AKR1C3 and upregulated by silencing AKR1C3. The ferroptosis-related gene AKR1C3 can be used as a diagnostic biomarker for asthma, particularly for MA, and regulates ferroptosis in BEAS-2B cells.

Establishing a Proteomics-Based Signature of AKR1C3-Related Genes for Predicting the Prognosis of Prostate Cancer

Aldo-keto reductase family 1 member C3 (AKR1C3) plays an important role in prostate cancer (PCa) progression, particularly in castration-resistant prostate cancer (CRPC). It is necessary to establish a genetic signature associated with AKR1C3 that can be used to predict the prognosis of PCa patients and provide important information for clinical treatment decisions. AKR1C3-related genes were identified via label-free quantitative proteomics of the AKR1C3-overexpressing LNCaP cell line. A risk model was constructed through the analysis of clinical data, PPI, and Cox-selected risk genes. Cox regression analysis, Kaplan-Meier (K-M) curves, and receiver operating characteristic (ROC) curves were used to verify the accuracy of the model, and two external datasets were used to verify the reliability of the results. Subsequently, the tumor microenvironment and drug sensitivity were explored. Moreover, the roles of AKR1C3 in the progression of PCa were verified in LNCaP cells. MTT, colony formation, and EdU assays were conducted to explore cell proliferation and drug sensitivity to enzalutamide. Migration and invasion abilities were measured using wound-healing and transwell assays, and qPCR was used to assess the expression levels of AR target genes and EMT genes. CDC20, SRSF3, UQCRH, INCENP, TIMM10, TIMM13, POLR2L, and NDUFAB1 were identified as AKR1C3-associated risk genes. These risk genes, established using the prognostic model, can effectively predict the recurrence status, immune microenvironment, and drug sensitivity of PCa. Tumor-infiltrating lymphocytes and several immune checkpoints that promote cancer progression were higher in high-risk groups. Furthermore, there was a close correlation between the sensitivity of PCa patients to bicalutamide and docetaxel and the expression levels of the eight risk genes. Moreover, through in vitro experiments, Western blotting confirmed that AKR1C3 enhanced SRSF3, CDC20, and INCENP expression. We found that PCa cells with a high expression of AKR1C3 have high proliferation ability and high migration ability and were insensitive to enzalutamide. AKR1C3-associated genes had a significant role in the process of PCa, immune responses, and drug sensitivity and offer the potential for a novel model for prognostic prediction in PCa.

Insulin-Induced AKR1C3 Induces Fatty Acid Synthase in a Model of Human PCOS Adipocytes.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women. In PCOS, insulin resistance and hyperandrogenism could drive the increased risk for cardiometabolic disease. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and is the predominant enzyme for potent androgen formation causing ligand-dependent androgen receptor (AR) activation. AR induces fatty acid synthase (FASN), a central enzyme for de novo lipogenesis. To investigate how insulin signaling induces AKR1C3 to promote lipid overload through induction of FASN, we used differentiated human Simpson-Golabi-Behmel syndrome adipocytes as a model for PCOS adipocytes. Induction of AKR1C3 and FASN was shown to be dependent on phosphoinositide 3-kinase/protein kinase B/ mammalian target of rapamycin/nuclear factor-erythroid 2-related factor 2 using pharmacological and genetic manipulation. FASN induction was shown to be AKR1C3 and AR dependent. Monofunctional AKR1C3 inhibitors, which competitively inhibit AKR1C3, did not block FASN induction, whereas bifunctional inhibitors, which competitively inhibit AKR1C3 and attenuate AR signaling by increasing AR degradation and ubiquitination, did suggesting a nonenzymatic role for AKR1C3 to stabilize AR. AKR1C3 and AR interacted as seen by co-immunoprecipitation, proximity ligation assay, and co-occupancy on FASN locus using chromatin immunoprecipitation-quantitative polymerase chain reaction assays in a ligand-dependent and ligand-independent manner. In the absence of androgens, bifunctional inhibitors prevented lipid droplet formation, whereas monofunctional inhibitors did not. We propose that AKR1C3 has 2 roles in PCOS: to catalyze potent androgen formation in adipocytes promoting hyperandrogenism and to induce FASN by stabilizing AR in the absence of androgens. AKR1C3 may be a therapeutic target for bifunctional inhibitors to reduce cardiometabolic disease in PCOS women.

Hyaluronic acid-FGF2-derived peptide bioconjugates for suppression of FGFR2 and AR simultaneously as an acne antagonist

Acne is a chronic skin condition that has serious consequences for mental and social well-being because it frequently occurs on the face. Several acne treatment approaches have commonly been used but have been hampered by side effects or weak activity. Thus, the investigation of the safety and efficacy of anti-acne compounds is of considerable medical importance. Herein, an endogenous peptide (P5) derived from fibroblast growth factors 2 (FGF2) was conjugated to the polysaccharide hyaluronic acid (HA) to generate the bioconjugate nanoparticle HA-P5, which suppresses fibroblast growth factor receptors (FGFRs) to significantly rehabilitate acne lesions and reduce sebum accumulation in vivo and in vitro. Moreover, our results show that HA-P5 inhibits both fibroblast growth factor receptor 2 (FGFR2) and androgen receptor (AR) signalling in SZ95 cells, reverses the acne-prone transcriptome, and decreases sebum secretion. Furthermore, the cosuppression mechanism revealed that HA-P5 blocks FGFR2 activation, as well as the YTH N6-methyladenosine RNA binding protein F3 (YTHDF3) downstream molecules, including an N6-methyladenosine (m6A) reader that facilitates AR translation. More importantly, a significant difference between HA-P5 and the commercial FGFR inhibitor AZD4547 is that HA-P5 does not trigger the overexpression of aldo-keto reductase family 1 member C3 (AKR1C3), which blocks acne treatment by catalyzing the synthesis of testosterone. Overall, we demonstrate that a polysaccharide-conjugated and naturally derived oligopeptide HA-P5 can alleviate acne and act as an optimal FGFR2 inhibitor and reveal that YTHDF3 plays a crucial role in signalling between FGFR2 and AR.Graphical

AKR1C2 Promotes Metastasis and Regulates the Molecular Features of Luminal Androgen Receptor Subtype in Triple Negative Breast Cancer Cells.

Patients with triple-negative breast cancer (TNBC) have an increased risk of distant metastasis compared to those with other subtypes. In this study, we aimed to identify the genes associated with distant metastasis in TNBC and their underlying mechanisms. We established patient-derived xenograft (PDX) models using surgically resected breast cancer tissues from 31 patients with TNBC. Among these, 15 patients subsequently developed distant metastases. Candidate metastasis-associated genes were identified using RNA sequencing. In vitro wound healing, proliferation, migration, and invasion assays and in vivo tumor xenograft and metastasis assays were performed to determine the functional importance of aldo-keto reductase family 1 member C2 (AKR1C2). Additionally, we used the METABRIC dataset to investigate the potential role of AKR1C2 in regulating TNBC subtypes and their downstream signaling activities. RNA sequencing of primary and PDX tumors showed that genes involved in steroid hormone biosynthesis, including AKR1C2, were significantly upregulated in patients who subsequently developed metastasis. In vitro and in vivo assays showed that silencing of AKR1C2 resulted in reduced cell proliferation, migration, invasion, tumor growth, and incidence of lung metastasis. AKR1C2 was upregulated in the luminal androgen receptor (LAR) subtype of TNBC in the METABRIC dataset, and AKR1C2 silencing resulted in the downregulation of LAR classifier genes in TNBC cell lines. The androgen receptor (AR) gene was a downstream mediator of AKR1C2-associated phenotypes in TNBC cells. AKR1C2 expression was associated with gene expression pathways that regulate AR expression, including JAK-STAT signaling or interleukin 6 (IL-6). The levels of phospho-signal transducer and activator of transcription and IL-6, along with secreted IL-6, were significantly downregulated in AKR1C2-silenced TNBC cells. Our data indicate that AKR1C2 is an important regulator of cancer growth and metastasis in TNBC and may be a critical determinant of LAR subtype features.

Function, drug resistance and prognostic effect of AKR1C2 in human cancer.

Aldo-keto reductases (ARKs), a group of reductases that rely on nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) to catalyze carbonyl, are widely found in various organisms, which play an important role in the physiological and pathological processes of human. Aldo-keto reductase family 1 member C2 (AKR1C2) as a member of the human ARKs family, can regulate steroid hormones and is abnormally expressed in many cancers. According to whether the tumor can be affected by hormones, we divide malignancies into hormone-dependent and hormone-independent types. Studies have shown that AKR1C2 is involved in regulating tumor invasion, migration, and other malignant phenotypes, eliminating reactive oxygen species (ROS), promoting chemotherapy resistance of tumor cells, and has prognostic value in some cancers. Here, we focus on the role and clinical significance of AKR1C2 in different types of tumors.

Computational modeling studies reveal the origin of the binding preference of 3-(3,4-di hydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids for AKR1C3 over its isoforms.

As a key regulator for hormone activity, human aldo-keto reductase family 1 member C3 (AKR1C3) plays crucial roles in the occurrence of various hormone-dependent or independent malignancies. It is a promising target for treating castration-resistant prostate cancer (CRPC). However, the development of AKR1C3 specific inhibitors remains challenging due to the high sequence similarity to its isoform AKR1C2. Here, we performed a combined in silico study to illuminate the inhibitory preference of 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids for AKR1C3 over AKR1C2, of which compound 38 can achieve up to 5000-fold anti-AKR1C3 selectivity. Our umbrella sampling (US) simulations together with end-point binding free energy calculation MM/GBSA uncover that the high inhibition selectivity originates from the different binding modes, namely "Inward" and "Outward," of this compound series in AKR1C3 and AKR1C2, respectively. In AKR1C3/38, the tetrahydroquinoline moiety of 38 is accommodated inside the SP1 pocket and interacts favorably with surrounding residues, while, in AKR1C2/38, the SP1 pocket is too small to hold the bulky tetrahydroquinoline group that instead moves out of the pocket with 38 transitioning from an "Inward" to an "Outward" state. Further 3D-QSAR and energy decomposition analyses suggest that SP1 in AKR1C3 prefers to bind with a rigid bicyclic moiety and the modification of the R3 group has important implication for the compound's activity. This work is the first attempt to elucidate the selectivity mechanism of inhibitors toward AKR1C3 at the atomic level, which is anticipated to propel the development of next-generation AKR1C3 inhibitors with enhanced efficacy and reduced "off-target" effect for CRPC therapy.

RF03 | PMON296 Insulin-induced AKR1C3 Metabolizes Classical and 11-oxygenated Androgens in a Model of PCOS Adipocytes

Abstract Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women. These patients commonly develop hyperandrogenism and this androgen excess (AE) is believed to drive the syndrome. PCOS women also characteristically develop insulin resistance (IR), which correlates with AE in PCOS women. Aldo-keto reductase family 1 member C3 (AKR1C3) catalyzes the conversion of peripheral androgens and is induced by insulin in adipocytes. Classically, AKR1C3 converts Δ4-androstene-3,17-dione (4AD) to testosterone (T) and 5α-androstane-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT). As a result, androgen conversion in adipocytes may contribute to the AE of PCOS women. Additionally, the 11-oxygenated androgens of adrenal origin were reported as the dominant androgens of PCOS and may be an alternative source of AE. AKR1C3 can also convert 11-ketoandrostene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-ketoandrostane-3,17-dione (11K-5AD) to 11-ketodihydrotestosterone (11K-DHT). T, DHT, 11K-T and 11K-DHT are potent agonists for the androgen receptor (AR). Therefore, overexpression of AKR1C3 by insulin could increase AR activation through production of both classical and 11-keto potent androgens. We hypothesize that AKR1C3 induced by insulin will produce 11K-T and 11K-DHT in a model of PCOS adipocytes. We developed a stable-isotope-dilution liquid chromatography high resolution mass spectrometric assay (SID-LC-HRMS) for the quantification of both classical and 11-oxygenated androgens in differentiated Simpson-Golabi-Behmel Syndrome adipocytes. Cells were treated with 4AD, 11K-4AD, or 11β-hydroxy-androstene-3,17-dione (11β-OH-4AD), the adrenal precursor to 11K-4AD. Androgens were derivatized with Girard P to enhance sensitivity and specificity. Analyte peaks were quantified using calibration curves of analyte to internal standard ratios versus pg of authentic standard. Our data suggests that 11β-OH-4AD is converted to 11K-4AD, which is then converted by insulin-induced AKR1C3 to 11K-T. The conversion of 11K-4AD to 11K-T was AKR1C3 dependent since a panel of AKR1C3 inhibitors blocked 11K-T formation. We found that 11K-T is deactivated to 11β-hydroxy-testosterone (11β-OH-T) and 11K-4AD can be back converted to 11β-OH-4AD by 11β-hydroxy steroid dehydrogenase type 1 (HSD11B1). This suggests that HSD11B1 protects the AR from overactivation by converting potent 11-keto androgens to their less potent 11β-hydroxy counterparts. Both classical and 11-oxygenated androgens may be sources of AE in PCOS by their intracrine formation in adipocytes. Our work elucidates the role of AKR1C3 in the formation of both classical and 11-keto potent androgens in a model of PCOS adipocytes and supports AKR1C3 as a potential therapeutic target in mitigating the AE of PCOS. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.