Pyrroline-5-carboxylate Reductase 1 Research Articles

PYCR1 promotes liver cancer cell growth and metastasis by regulating IRS1 expression through lactylation modification.

Liver cancer (LC) is among the deadliest cancers worldwide, with existing treatments showing limited efficacy. This study aimed to elucidate the role and underlying mechanisms of pyrroline-5-carboxylate reductase 1 (PYCR1) as a potential therapeutic target in LC. Immunohistochemistry and Western blot were used to analyse the expression of PYCR1 in LC cells and tissues. EdU assays, colony-forming assays, scratch wound healing assays, Transwell assays, nude mouse xenograft models and nude mouse lung metastasis models were used to detect the growth and metastasis abilities of LC cells. Transcriptome sequencing was used to search for downstream target genes regulated by PYCR1, and metabolomics was used to identify the downstream metabolites regulated by PYCR1. ChIP assays were used to analyse the enrichment of H3K18 lactylation in the IRS1 promoter region. We found that the expression of PYCR1 was significantly increased in HCC and that this high expression was associated with poor prognosis in HCC patients. Knockout or inhibition of PYCR1 inhibited HCC cell proliferation, migration and invasion both in vivo and in vitro. In addition, we revealed that knocking out or inhibiting PYCR1 could inhibit glycolysis in HCC cells and reduce H3K18 lactylation of the IRS1 histone, thereby inhibiting IRS1 expression. Our findings identify PYCR1 as a pivotal regulator of LC progression that influences tumour cell metabolism and gene expression. By demonstrating the potential of targeting PYCR1 to inhibit LC cell proliferation and metastasis, this study identified PYCR1 as a promising therapeutic target for LC. Pyrroline-5-carboxylate reductase 1 (PYCR1) promotes the proliferation and metastasis of liver cancer (LC) cells. The expression of PYCR1 in LC is regulated by DNA methylation. Knocking down or inhibiting PYCR1 inhibits glycolysis as well as the PI3K/AKT/mTOR and MAPK/ERK pathways in LC cells. PYCR1 regulates the transcriptional activity of IRS1 by affecting H3K18 lactylation in its promoter region.

Crosstalk between FTH1 and PYCR1 dysregulates proline metabolism and mediates cell growth in KRAS-mutant pancreatic cancer cells.

Ferritin, comprising heavy (FTH1) and light (FTL) chains, is the main iron storage protein, and pancreatic cancer patients exhibit elevated serum ferritin levels. Specifically, higher ferritin levels are correlated with poorer pancreatic ductal adenocarcinoma (PDAC) prognosis; however, the underlying mechanism and metabolic programming of ferritin involved in KRAS-mutant PDAC progression remain unclear. Here, we observed a direct correlation between FTH1 expression and cell viability and clonogenicity in KRAS-mutant PDAC cell lines as well as with in vivo tumor growth through the control of proline metabolism. Our investigation highlights the intricate relationship between FTH1 and pyrroline-5-carboxylate reductase 1 (PYCR1), a crucial mitochondrial enzyme facilitating the glutamate-to-proline conversion, underscoring its impact on proline metabolic imbalance in KRAS-mutant PDAC. This regulation is further reversed by miR-5000-3p, whose dysregulation results in the disruption of proline metabolism, thereby accentuating the progression of KRAS-mutant PDAC. Additionally, our study demonstrated that deferasirox, an oral iron chelator, significantly diminishes cell viability and tumor growth in KRAS-mutant PDAC by targeting FTH1-mediated pathways and altering the PYCR1/PRODH expression ratio. These findings underscore the novel role of FTH1 in proline metabolism and its potential as a target for PDAC therapy development.

Confirming the enzymatic activity and neurodevelopmental trajectory of PYCR1 mutation in one child with autosomal-recessive cutis laxa type 2

Autosomal-recessive cutis laxa type 2 (ARCL2) is a rare genetic disorder caused by pyrroline-5-carboxylate reductase 1 (PYCR1) mutations and characterized by loose and sagging skin, typical facial features, intrauterine growth retardation, and developmental delay. To study the effect of PYCR1 mutations on protein function and clinical features, we identified a homozygous missense mutation c.559G > A (p.Ala187Thr) in PYCR1 in a Chinese child with typical clinical features, especially severe developmental delays. The three-dimensional (3D) model showed the modification of the hydrogen bonds produce a misfolding in the mutant PYCR1 protein. Mutagenesis and enzyme assay study revealed decreased activity of the mutant protein in vitro, indicating that this mutation impairs PYCR1 function. Our findings confirmed abnormal enzymatic activity and neurodevelopmental trajectory of this PYCR1 mutation.

PYCR1 expresses in cancer-associated fibroblasts and accelerates the progression of C6 glioblastoma.

Cancer-associated fibroblasts (CAFs) play important roles in tumor microenvironments. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a potential cancer therapy target. This study aimed to explore the expression of PYCR1 in glioma-associated CAFs and analyze the effects of PYCR1 expression in CAFs on the proliferation of C6 glioma. A rat glioma model was induced by injecting C6 cells in the right caudate putamen via a microliter syringe. After 14 days, tumor tissues were collected, and the levels of COL1A1 and PYCR1 were measured by immunohistochemistry. The colocalization of fibroblast activation protein α (FAP) and PYCR1 in tissues was measured by double-immunofluorescence. The CAFs were labeled by FAP and isolated from the tumor tissues using a fluorescence-activated cell sorting (FACS) machine. The isolated CAFs were further separated into CAFs with different PYCR1 expressions using the FACS machine. CAFs with different PYCR1 expressions were respectively cocultured with C6 cells or MUVECs for 48h using a Transwell permeable support. The invasion and proliferation of C6 cells were measured using a Transwell assay and colony formation assay, and the angiogenesis of MUVECs was measured using a Tube formation assay. The expression of COL1A1 and PYCR1 proteins in C6 cells and VEGF-A and EGF proteins in MUVECs was measured by western blotting. PYCR1 silencing in C6 cells was induced by PYCR1 siRNA transfection, the effects of which on the proliferation of C6 cells were measured using a wound healing assay, a Transwell assay, and western blotting. The PYCR1 and COL1A1 upregulation co-occurred in the rat glioma, and PYCR1 was expressed in CAFs. The CAF coculture enhanced the invasion and proliferation of C6 cells and the angiogenesis of MUVECs. Meanwhile, the levels of COL1A1 protein in C6 cells, and the levels of VEGF-A and EGF proteins in MUVECs were increased after CAF coculture. Moreover, the effects of CAF coculture were increased with PYCR1 expression in the CAF. Silencing PYCR1 suppressed the migration and invasion of C6 cells, and decreased the levels of COL1A1 and VEGF-A proteins in C6 cells. PYCR1 is expressed in glioma-associated CAFs, and promotes the proliferation of C6 cells and angiogenesis of MUVECs, suggesting that targeting PYCR1 may be a therapeutic strategy for glioma.

Metabolic characterization of sphere-derived prostate cancer stem cells reveals aberrant urea cycle in stemness maintenance.

Alteration of cell metabolism is one of the essential characteristics of tumor growth. Cancer stem cells (CSCs) are the initiating cells of tumorigenesis, proliferation, recurrence, and other processes, and play an important role in therapeutic resistance and metastasis. Thus, identification of the metabolic profiles in prostate cancer stem cells (PCSCs) is critical to understanding prostate cancer progression. Using untargeted metabolomics and lipidomics methods, we show distinct metabolic differences between prostate cancer cells and PCSCs. Urea cycle is the most significantly altered metabolic pathway in PCSCs, the key metabolites arginine and proline are evidently elevated. Proline promotes cancer stem-like characteristics via the JAK2/STAT3 signaling pathway. Meanwhile, the enzyme pyrroline-5-carboxylate reductase 1 (PYCR1), which catalyzes the conversion of pyrroline-5-carboxylic acid to proline, is highly expressed in PCSCs, and the inhibition of PYCR1 suppresses the stem-like characteristics of prostate cancer cells and tumor growth. In addition, carnitine and free fatty acid levels are significantly increased, indicating reprogramming of fatty acid metabolism in PCSCs. Reduced sphingolipid levels and increased triglyceride levels are also observed. Collectively, our data illustrate the comprehensive landscape of the metabolic reprogramming of PCSCs and provide potential therapeutic strategies for prostate cancer.

PYCR1 regulates TRAIL‑resistance in non‑small cell lung cancer cells by regulating the redistribution of death receptors.

Although recombinant human TNF-related apoptosis-inducing ligand (TRAIL) protein exhibits antitumor activity in a number of lung and liver cancer cells and tumor-bearing animals, TRAIL resistance has substantially restricted its clinical application. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a key enzyme in the regulation of proline synthesis. PYCR1 is highly expressed in various types of malignant tumor, in which it has been implicated in 5-fluorouracil resistance. However, the possible relationship between PYCR1 and TRAIL resistance remains unclear. In the present study, both reverse transcription-quantitative PCR and western blotting were performed. The results indicated that H1299 cells had higher PYCR1 expression levels and were less sensitive to TRAIL compared with the TRAIL-sensitive cell line, H460. PYCR1 knockdown in H1299 cells increased TRAIL sensitivity, increased the localization of death receptors (DRs) on the cell surface and activated Caspase-3/8. By contrast, overexpression of PYCR1 in H1299 cells decreased TRAIL sensitivity, reduced the distribution of DRs on the cell surface and suppressed the activation of Caspase-3/8. Taken together, these results suggested that PYCR1 promoted TRAIL resistance in the non-small cell lung cancer cell line, H1299, by preventing redistribution of DRs to the plasma membrane. This in turn inhibited TRAIL-mediated cell apoptosis by reducing the activation of Caspase-3/8.

Pro-tumorigenic activity of PYCR1 in gastric cancer through regulating the PI3K/AKT signaling

BackgroundThe primary objective of this investigation was to assess the impact of pyrroline-5-carboxylate reductase 1 (PYCR1) on the progression of gastric cancer (GC), specifically focusing on tumor growth and metastatic potential. MethodsSurgical specimens from patients with different stages of GC were assayed for PYCR1 expression using immunohistochemistry. PYCR1 expression was manipulated by depletion or overexpression approaches in GC cells, and these cells were applied to explore the functional roles of PYCR1. Expression of apoptosis– and metastasis–related markers was quantified through quantitative real-time PCR and Western blot. ResultsHigher PYCR1 expression was ascertained in surgical specimens from patients with GC as compared to noncancerous adjacent tissues. Additionally, PYCR1 overexpression in GC tissues was linked to adverse clinical outcomes. The depletion of PYCR1 in GC cells resulted in a pronounced reduction in proliferation, the induction of apoptosis, and the attenuation of invasion and metastasis. Conversely, its ectopic expression notably augmented proliferation, restricted apoptosis, and stimulated invasion and metastasis. In addition, the knockdown of PYCR1 resulted in a significant elevation in the activation of caspase 3, a key protein involved in apoptosis. This depletion also led to a decrease in the activation or expression of proteins associated with metastasis, such as phosphorylated (p)-phosphatidylinositol 3-kinase (PI3K), p-AKT serine/threonine kinase (AKT), and snail family transcriptional repressor 1 (Snail). Additionally, it resulted in an upregulation of E-cadherin expression. Conversely, the overexpression of PYCR1 notably increased the levels of p-PI3K, p-AKT, and Snail, while simultaneously reducing E-cadherin expression. ConclusionPYCR1, by activating PI3K/AKT signaling, assumes a crucial role in governing malignant characteristics of GC cells, including proliferation, apoptosis, and metastasis. These findings underscore the promising potential of PYCR1 as a diagnostic biomarker and a target for tailored therapeutic interventions in patients with GC.

P003 Metabolic Reprogramming in Intestinal Fibrosis: Integrating Metabolomics, Single-Cell Transcriptomics and Isotope Tracing

Abstract Background This study aims to systematically elucidate the metabolic landscape of intestinal fibrosis in Crohn's disease, revealing the key reprogrammed metabolic pathways in the fibrotic microenvironment. Methods We collected normal and fibrosis tissues from 122 Crohn's disease patients undergoing surgery for fibrosis-induced intestinal obstruction, and established DSS/TNBS-induced murine intestinal fibrosis model. To measure metabolites level in normal and fibrotic tissues, untargeted metabolomics was performed. C13-labeled glutamine was used for metabolic tracing to delineate metabolic fluxes in vitro and in vivo. We performed integration analysis by merging metabolomics and metabolic tracing data with established single-cell transcriptomic profiles. Isolation and further validation of human intestinal stromal cells were carried out, and gene expression was assessed using qRT-PCR and immunofluorescence. Results Significant metabolic heterogeneity was observed between normal and fibrotic tissues, with differential metabolites enriched in glucose and amino acid metabolism. Notably, intermediates of glycolysis were significantly increased, while tricarboxylic acid (TCA) cycle intermediates were decreased in fibrotic regions (p<0.05). Amino acids were broadly downregulated in fibrosis, whereas glutamate and glutamine were highly enriched (p<0.05). Mechanistic insights into these changes were provided by isotope tracing, revealing enhanced glycolysis and weakened TCA flux attributed to transition from TCA intermediate α-KG to glutamate. Moreover, catabolism of glutamine to glutamate was enhanced. Glutamate from above pathways converted to proline, which is the ingredient of extracellular matrix (ECM). Single-cell transcriptomic analysis indicated a high enrichment of genes related to glutamine metabolism in stromal cells, with significant upregulation of enzymes such as GLS (Glutaminase) and PYCR1 (Pyrroline-5-carboxylate Reductase 1) involved in glutamine and proline synthesis. Immunofluorescence and qRT-PCR confirmed the upregulation of GLS and PYCR1. Further experiments using primary stromal cells and murine models demonstrated that inhibiting GLS activity reduced ECM synthesis, alleviating intestinal fibrosis. Conclusion In intestinal fibrosis, glycolysis and glutamine catabolism enhanced. Stromal cells utilized the reprogrammed metabolic pathway to synthesize glutamate, thereby promoting pathological ECM anabolism. Targeting the key enzyme GLS represent a potential therapeutic strategy for intestinal fibrosis.

PYCR1, BANF1, and STARD8 Expression in Gastric Carcinoma: A Clinicopathologic, Prognostic, and Immunohistochemical Study.

It will be important to understand the molecular pathways of gastric cancer (GC) occurrence and progression, thus detecting predictive and prognostic biomarkers of GC. Pyrroline-5-carboxylate reductase 1 (PYCR1) was upregulated in many cancers, suggesting its possible roles in carcinogenesis and tumor metastases. Barrier-of-autointegration factor 1 (BANF1) is a protein family that plays essential roles in maintaining the integrity of an intact cellular genome. Rho-GTPs are molecular switches that control many signal transduction pathways in normal cells, including 3 subgroups from 1 to 3 (DLC1-3). DLC-3, known as StAR-related lipid transfer domain protein 8 (STARD8), and its role in cancers were not sufficiently studied. The study aimed to investigate the significance of PYCR1, BANF1, and STARD8 protein expression in GC tissues and normal gastric mucosa retrieved from patients with GC to detect prognostic roles of expression. Specimens were collected from 100 patients with gastric carcinoma. After the application of the inclusion criteria of the study, we prepared 100 paraffin blocks from samples of the 100 included patients; each block included samples from gastric carcinoma and adjacent non-neoplastic gastric mucosa. We assessed the expression of PYCR1, BANF1, and STARD8 using immunohistochemistry in all studied samples. We followed patients for the detection of disease progression and survival rates. We correlate PYCR1, BANF1, and STARD8 expression with clinical, pathologic, and prognostic parameters. Overexpression of PYCR1 and BANF1 and decreased expression of STARD8 was found in gastric carcinoma tissues than adjacent non-neoplastic gastric mucosa ( P <0.001), and was positively associated with high grade ( P =0.006), depth of tumor invasion, presence of lymph nodes metastases and advanced stage ( P =0.001), high incidence of GC progression, recurrence, unfavorable disease-free survival ( P =0.003) and unfavorable overall survival rates ( P <0.001). Thus, it was revealed that; in univariate and multivariate analyses, levels of PYCR1, BANF1, and STARD8 are associated with the overall survival rate of GC patients. We showed that overexpression of PYCR1 and BANF1 and decreased expression of STARD8 in GC tissues was associated with poor prognosis and GC progression.

Proline is increased in allergic asthma and promotes airway remodeling.

Proline and its synthesis enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) are implicated in epithelial-mesenchymal transition (EMT), yet how proline and PYCR1 function in allergic asthmatic airway remodeling via EMT has not yet been addressed to our knowledge. In the present study, increased levels of plasma proline and PYCR1 were observed in patients with asthma. Similarly, proline and PYCR1 in lung tissues were high in a murine allergic asthma model induced by house dust mites (HDMs). Pycr1 knockout decreased proline in lung tissues, with reduced airway remodeling and EMT. Mechanistically, loss of Pycr1 restrained HDM-induced EMT by modulating mitochondrial fission, metabolic reprogramming, and the AKT/mTORC1 and WNT3a/β-catenin signaling pathways in airway epithelial cells. Therapeutic inhibition of PYCR1 in wild-type mice disrupted HDM-induced airway inflammation and remodeling. Deprivation of exogenous proline relieved HDM-induced airway remodeling to some extent. Collectively, this study illuminates that proline and PYCR1 involved with airway remodeling in allergic asthma could be viable targets for asthma treatment.

Effects of PYCR1 on prognosis and immunotherapy plus tyrosine kinase inhibition responsiveness in metastatic renal cell carcinoma patients

BackgroundImmunotherapy plus tyrosine kinase inhibitor (IO-TKI) has become the first-line management for metastatic renal cell carcinoma (RCC), despite the absence of biomarkers. Recently, pyrroline-5-carboxylate reductase 1 (PYCR1) and proline metabolism have been reported regulatory roles in the anti-tumor response. MethodsThere were three cohorts enrolled: two from our institution (ZS-MRCC and ZS-HRRCC) and one from a clinical trial (JAVELIN-101). The PYCR1expression in each sample was evaluated by RNA sequencing. Flow cytometry and immunohistochemistry were performed to assess immune infiltration. Single-cell RNA-seq (scRNA-seq) data was used for cluster analysis of T cells and macrophages. Primary endpoints were set as response and progression-free survival (PFS). ResultsPatients in the low-PYCR1 group had greater objective response rate (52.2% vs 18.2%) and longer PFS in both cohorts (ZS-MRCC cohort, P=0.01, HR=2.80; JAVELIN-101 cohort, P<0.001, HR=1.85). In responders, PYCR1 expression was decreased (P<0.05). In the high PYCR1 group, CD8+ T cells exhibited an exhausted phenotype with decreased GZMB (Spearman's ρ=-0.36, P=0.02). scRNA-seq revealed tissue-resident memory T (Trm) (P<0.05) and tissue-resident macrophage (P<0.01) were decreased in samples with high PYCR1 expression. A machine learning score was further built by random forest, involving PYCR1 and Trm markers. Only in the subgroup with the lower RFscore did IO+TKI show a favorable outcome, compared to TKI monotherapy. ConclusionsImmunosuppression and IO+TKI resistance were correlated with high PYCR1 expression. T cell exhaustion and dysfunction were also related with the expression of PYCR1. PYCR1 has the potential to be employed as a biomarker to discriminate between IO+TKI and TKI monotherapy as the optimal patient treatment strategy.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Antisense lncRNA-RP11-498C9.13 promotes bladder cancer progression by enhancing reactive oxygen species-induced mitophagy.

Urinary system's most prevalent malignant tumor is bladder cancer. The enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) has pro-tumorigenic characteristics. In the present study, the upstream and downstream regulatory mechanisms of PYCR1 in bladder cancer were investigated. The relationship between the expression of PYCR1 in bladder cancer and its prognosis was analyzed using a bioinformatics technique. Plasmid transfection and small interfering RNA were utilized to overexpress and silence genes, respectively. Utilizing MTT, colony formation, EdU, and transwell assays, the proliferation and invasiveness of bladder cancer cells were evaluated. Employing an RNA pull-down experiment and RNA immunoprecipitation, the relationship between RNAs was analyzed. Fluorescence in situ hybridization, immunohistochemistry, and western blotting were used to detect protein expression and localization. Flow cytometry was used to identify reactive species (ROS) expression in cells. Mitophagy was detected using immunofluorescence. PYCR1 was highly expressed in bladder cancer tissue and was related with a poor prognosis for the patient. By binding to PYCR1, the antisense RNA lncRNA-RP11-498C9.13 prevented the degradation of PYCR1 and promoted its production. Down-regulation of lncRNA-RP11-498C9.13 and PYCR1 inhibited the proliferation and invasiveness of bladder cancer cells and decreased tumorigenesis. In addition, it was found that the lncRNA-RP11-498C9.13/PYCR1 axis promoted ROS generation and induced mitophagy in bladder cancer cells. We demonstrated that lncRNA-RP11-498C9.13 promoted bladder cancer tumorigenesis by stabilizing the mRNA of PYCR1 and promoted ROS-induced mitophagy. The lncRNA-RP11-498C9.13/PYCR1/mitophagy axis was anticipated to be a significant therapeutic target for bladder cancer.

Exosomes from PYCR1 knockdown bone marrow mesenchymal stem inhibits aerobic glycolysis and the growth of bladder cancer cells via regulation of the EGFR/PI3K/AKT pathway.

Bladder cancer (BC) is a heterogeneous disease, and pyrroline‑5‑carboxylate reductase 1 (PYCR1) can promote the proliferation and invasion of BC cells and accelerate BC progression. In the present study, si‑PYCR1 was loaded into bone marrow mesenchymal stem cell (BMSC)‑derived exosomes (Exos) in BC. First, PYCR1 levels in BC tissues/cells were assessed, and cell proliferation, invasion, and migration were evaluated. Aerobic glycolysis levels (glucose uptake, lactate production, ATP production, and the expression of relevant enzymes) and the EGFR/PI3K/AKT pathway phosphorylation levels were determined. PYCR1‑EGFR interactions were examined by co‑immunoprecipitation experiments. RT4 cells transfected with oe‑PYCR1 were treated with EGFR inhibitor CL‑387785. Exos were loaded with si‑PYCR1 and identified, followed by an assessment of their effects on aerobic glycolysis and malignant cell behaviors. Nude mouse models of xenograft tumors were established by injecting mice with Exo‑si‑PYCR1 and Exo‑si‑PYCR1. PYCR1 was upregulated in BC cells, with the highest expression observed in T24 cells and the lowest expression in RT4 cells. Following PYCR1 knockdown, the malignant behaviors of T24 cells and aerobic glycolysis were decreased, while PYCR1 overexpression in RT4 cells averted these trends. PYCR1 interacted with EGFR, and CL‑387785 inhibited the EGFR/PI3K/AKT pathway and attenuated the effects of PYCR1 overexpression on RT4 cells but had no effect on PYCR1 expression. Exo‑si‑PYCR1 showed stronger inhibitory effects on aerobic glycolysis and on the malignant behaviors of T24 cells than si‑PYCR1. Exo‑si‑PYCR1 blocked xenograft tumor growth and had good biocompatibility. Briefly, PYCR1 knocking loaded by BMSC‑derived Exos suppressed aerobic glycolysis and BC growth via the PI3K/AKT pathway by binding to EGFR.

Meta-omics profiling of the gut-lung axis illuminates metabolic networks and host-microbial interactions associated with elevated lung elastance in a murine model of obese allergic asthma.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

PYCR1 promotes the malignant progression of lung cancer through the JAK-STAT3 signaling pathway via PRODH-dependent glutamine synthesize

BackgroundLung cancer is a serious threat to human life. It is of great significance to elucidate the pathogenesis of lung cancer and search for new markers. This study evaluate the clinical value of pyrroline-5-carboxylate reductase 1 (PYCR1) and explore its role and mechanisms in the malignant progression of lung cancer. MethodsPYCR1 expression and its relationship with prognosis were analyzed using a bioinformatics database. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were utilized to examine the expression of PYCR1 in lung cancer tissues and peripheral blood. PYCR1-overexpressing lung cancer cells were constructed, then the cell proliferative, migration, and invasion ability was examined by the MTT and Transwell assays. siRNA against PRODH and STAT3 inhibitor sttatic was used to further elucidate the underlying mechanisms. Luciferase and CHIP assays were carried out for validate the how PYCR1 regulated PD-L1 expression via STAT3. Xenograft experiment was performed to determine the role of PYCR1 in vivo. ResultsDatabase analysis showed that PYCR1 expression was significantly increased in lung cancer tissues, and its high expression predicted poor prognosis. Lung cancer tissue and peripheral blood of patients showed obviously increased PYCR1 expression, and the sensitivity and specificity of serum PYCR1 in the diagnosis of lung cancer were 75.7% and 60%, respectively. PYCR1 overexpression enhanced the proliferative, migration, and invasion abilities of lung cancer cells. Both PRODH silence and stattic effectively attenuated the function of PYCR1. Animal experiment and IHC data indicated that PYCR1 could activated STAT3 phosphorylation and PD-L1, as well as suppressed T cell infiltration in lung cancer. Finally, we also validated that PYCR1 promoted PD-L1 transcription by elevating STAT3 binding to the gene promoter. ConclusionPYCR1 has certain value in the diagnosis and prognosis of lung cancer. Moreover, through regulating JAK-STAT3 signaling pathway, PYCR1 significantly participated in process of lung cancer progression via the metabolism link between proline and glutamine, indicating that PYCR1 might be also a novel therapeutic target.

Targeting IGF1R signaling enhances the sensitivity of cisplatin by inhibiting proline and arginine metabolism in oesophageal squamous cell carcinoma under hypoxia

BackgroundCisplatin (DDP)-based chemotherapy is commonly adopted as the first-line treatment for patients with oesophageal squamous cell carcinoma (OSCC), but the high rate of drug resistance limits its clinical application and the underlying mechanisms at play remain unclear. The aims of this study were to elucidate the role of abnormal signal transmission and metabolism in the chemoresistance of OSCC under hypoxia and to identify targeted drugs that enhance the sensitivity of DDP chemotherapy.MethodsUpregulated genes in OSCC were determined by RNA sequencing (RNA-seq), the Cancer Genome Atlas (TCGA) database, immunohistochemistry (IHC), real-time quantitative PCR (RT-qPCR), and western blotting (WB). The clinicopathological significance of insulin-like growth factor-I receptor (IGF1R), argininosuccinate synthetase 1 (ASS1), and pyrroline-5-carboxylate reductase 1 (PYCR1) in OSCC was analysed using tissue micriarray (TMA). Metabolic abnormalities were determined by untargeted metabolomics analysis. The DDP-resistance role of IGF1R, ASS1, and PYCR1 in OSCC was investigated in vitro and in vivo.ResultsGenerally, tumour cells exist in a hypoxic microenvironment. By genomic profiling, we determined that IGF1R, as a receptor tyrosine kinase (RTK), was upregulated in OSCC under low-oxygen conditions. Clinically, enhanced IGF1R expression was associated with higher tumour stages and a poorer prognosis in OSCC patients, and its inhibitor, linsitinib, showed synergistic effects with DDP therapy in vivo and in vitro. Since oxygen-deprivation frequently lead to metabolic reprogramming, we further learned via metabolomics analysis that abnormal IGF1R pathways promoted the expression of metabolic enzymes ASS1 and PYCR1 by the transcriptional activity of c-MYC. In detail, enhanced expression of ASS1 promotes arginine metabolism for biological anabolism, whereas PYCR1 activates proline metabolism for redox balance, which maintains the proliferation ability of OSCC cells during DDP treatment under hypoxic conditions.ConclusionEnhanced expression of ASS1 and PYCR1 via IGF1R pathways rewired arginine and proline metabolism, promoting DDP resistance in OSCC under hypoxia. Linsitinib targeting IGF1R signaling may lead to promising combination therapy options for OSCC patients with DDP resistance.

Evaluation of aristolochic acid Ι nephrotoxicity in mice via 1H NMR quantitative metabolomics and network pharmacology approaches.

Although many studies have shown that herbs containing aristolochic acids can treat various human diseases, AAΙ in particular has been implicated as a nephrotoxic agent. Here, we detail the nephrotoxic effect of AAΙ via an approach that integrated 1H NMR-based metabonomics and network pharmacology. Our findings revealed renal injury in mice after the administration of AAΙ. Metabolomic data confirmed significant differences among the renal metabolic profiles of control and model groups, with significant reductions in 12 differential metabolites relevant to 23 metabolic pathways. Among them, there were seven important metabolic pathways: arginine and proline metabolism; glycine, serine, and threonine metabolism; taurine and hypotaurine metabolism; ascorbate and aldehyde glycolate metabolism; pentose and glucosinolate interconversion; alanine, aspartate, and glutamate metabolism; and glyoxylate and dicarboxylic acid metabolism. Relevant genes, namely, nitric oxide synthase 1 (NOS1), pyrroline-5-carboxylate reductase 1 (PYCR1), nitric oxide synthase 3 (NOS3) and glutamic oxaloacetic transaminase 2 (GOT2), were highlighted via network pharmacology and molecular docking techniques. Quantitative real-time PCR findings revealed that AAI administration significantly downregulated GOT2 and NOS3 and significantly upregulated NOS1 and PYCR1 expression and thus influenced the metabolism of arginine and proline. This work provides a meaningful insight for the mechanism of AAΙ renal injury.

LINC01123 acts as an oncogenic driver in lung adenocarcinoma by regulating the miR-4766-5p/PYCR1 axis.

Lung adenocarcinoma remains one of the most significant threats to human life as it involves multiple etiologies, including alteration of oncogenes or tumor-inhibitory genes. Long non-coding RNAs (lncRNAs) have been reported to have both cancer promoting and cancer inhibiting effects. In this work, we investigated the function and mechanism of lncRNA LINC01123 in lung adenocarcinoma. The expression of LINC01123, miR-4766-5p, and PYCR1 (pyrroline-5-carboxylate reductase 1) mRNA was analyzed by RT-qPCR. The protein expression levels of PYCR1 and the apoptosis-related proteins (Bax and Bcl-2) were determined by western blotting. Cell proliferation and migration were determined by CCK-8 and wound-healing assays, respectively. Tumor growth in nude mice and Ki67 immunohistochemical staining were used to determine the in vivo role of LINC01123. The putative binding relationships miR-4766-5p has with LINC01123 and PYCR1, which had been identified by analysis of public databases, were validated through RIP and dual-luciferase reporter assays. LINC01123 and PYCR1 overexpression and miR-4766-5p downregulation were shown to occur in lung adenocarcinoma samples. LINC01123 depletion repressed lung adenocarcinoma cell growth and migration and blocked the development of solid tumors in an animal model. Moreover, LINC01123 bound directly to miR-4766-5p, the downregulation of which attenuated the anticancer effects of LINC01123 depletion in lung adenocarcinoma cells. MiR-4766-5p directly targeted downstream PYCR1 to suppress PYCR1 expression. The repressive effects of PYCR1 knockdown on the migration and proliferation of lung adenocarcinoma cells were also partly abolished by miR-4766-5p downregulation. Downregulation of LINC01123 represses lung adenocarcinoma progression. This suggests that LINC01123 functions as an oncogenic driver in lung adenocarcinoma by controlling the miR-4766-5p/PYCR1 axis.

LINC00511 aggravates the malignancy of lung adenocarcinoma through sponging microRNA miR-4739 to regulate pyrroline-5-carboxylate reductase 1 expression.

Long non-coding RNA LINC00511 is known to exacerbate lung adenocarcinoma (LUAD) progression. However, the specific mechanism by which LINC00511 affects LUAD progression has not been investigated as yet, and we aimed to elucidate the same in the present study. The expression levels of LINC00511, microRNA miR-4739, and pyrroline-5-carboxylate reductase 1 (PYCR1) were determined by quantitative reverse transcription PCR and Western blotting. The Cell Counting Kit-8 and bromodeoxyuridine assays were used to evaluate cell proliferation. Apoptosis was evaluated by flow cytometry, and Bax and Bcl-2 protein levels were determined by western blotting. Cell migration was assessed using transwell assay. The interaction between LINC00511, miR-4739, and PYCR1 was analyzed using luciferase, RNA immunoprecipitation, and RNA pull-down assays. The expression levels of LINC00511 and PYCR1 in LUAD were downregulated, whereas that of miR-4739 was upregulated. Functional studies showed that knockdown of LINC00511 or PYCR1 suppressed the proliferation and migration of LUAD cells, and promoted apoptosis. On the contrary, knockdown of miR-4739 had tumor-promoting effects. Mechanistically, LINC00511 prevented the miR-4739 led inhibition of PYCR1, resulting in PYCR1 overexpression. This study demonstrates for the first time that LINC00511 aggravates the malignancy of LUAD by sponging miR-4739 to upregulate PYCR1 expression.