Novel Fragment Inhibitors of PYCR1 from Docking-Guided X-ray Crystallography.

The proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate (P5C) reductase 1 (PYCR1) is one of the most consistently upregulated enzymes across multiple cancer types and central to the metabolic rewiring of cancer cells. Herein, we describe a fragment-based, structure-first approach to the discovery of PYCR1 inhibitors. Thirty-seven fragment-like carboxylic acids in the molecular weight range of 143-289 Da were selected from docking and then screened using X-ray crystallography as the primary assay. Strong electron density was observed for eight compounds, corresponding to a crystallographic hit rate of 22%. The fragments are novel compared to existing proline analog inhibitors in that they block both the P5C substrate pocket and the NAD(P)H binding site. Four hits showed inhibition of PYCR1 in kinetic assays, and one has lower apparent IC50 than the current best proline analog inhibitor. These results show proof-of-concept for our inhibitor discovery approach and provide a basis for fragment-to-lead optimization.

PYCR2, induced by c-Myc, promotes the invasiveness and metastasis of breast cancer by activating AKT signalling pathway.

BACKGROUND: Pyrroline-5-carboxylate reductase 2 (PYCR2) expression is aberrantly upregulated in colon cancer. However, the functions and underlying mechanisms of PYCR2 in breast cancer remain elusive. The primary objective of the present study was to elucidate the function of PYCR2 in breast cancer and investigate whether PYCR2 may be transcriptionally regulated by c-Myc to activate the AKT signaling pathway. METHODS: Immunohistochemical analysis was performed to examine the expression of PYCR2 in breast cancer and adjacent non-cancerous tissues. Western blot and RT-qPCR were utilized to detect PYCR2 expression in breast cancer cells. Cellular functionalities were evaluated through Transwell assays in vitro and lung metastasis formation assays in vivo. Moreover, the impact of PYCR2 on the activation of AKT signaling was determined through western blot and immunohistochemistry analysis. The transcriptional regulation of PYCR2 expression by c-Myc was evaluated via both western blot analysis and luciferase gene reporter assay. RESULTS: PYCR2 overexpression was noted in breast cancer. Silencing PYCR2 expression attenuated the invasive and metastatic abilities of breast cancer cells. Furthermore, the activation of the AKT signaling pathway is indispensable for the promotion of invasion and metastasis mediated by PYCR2. Lastly, the binding of c-Myc to the promoter sequence of PYCR2 resulted in the upregulation of PYCR2 transcription. CONCLUSION: Taken together, these results indicate that PYCR2 is transcriptionally regulated by c-Myc and promotes invasion and metastasis in breast cancer through the activation of the AKT pathway.

PYCR2 promotes growth and aerobic glycolysis in human liver cancer by regulating the AKT signaling pathway.

Hepatocellular carcinoma (HCC) is the world's third most fatal cancer. Because metabolic rewiring is a hallmark of HCC, studies into the causes of aberrant glycolysis could provide insight into novel HCC therapeutic strategies. Pyrroline-5-carboxylate reductase 2 (PYCR2), a key enzyme of proline synthesis, has previously been found to play vital roles in various malignancies regarding amino acid metabolism and oxidative stress response. Our study investigated the mechanistic function of PYCR2 in HCC. We used Gene Expression Profiling Interactive Analysis to perform bioinformatics analysis of PYCR2 expression and survival in human HCC patients based on the Cancer Genome Atlas database. The function of PYCR2 in cell viability and glycolysis was assessed using CCK-8 and ECAR assays. Transducing shRNA or overexpression vectors into the HCC cell line altered the expression status of PYCR2. PYCR2 expression was validated using quantitative real-time PCR and Western blot. In mouse xenograft models, the role of PYCR2 in HCC tumor formation was confirmed. PYCR2 was overexpressed in human HCC tumor tissue and was associated with a poor prognosis. The functional assay revealed that silencing PYCR2 inhibited cell viability, glycolysis, and AKT activation. Furthermore, the xenograft experiment demonstrated that silencing PYCR2 significantly inhibited tumor growth and Ki67 expression. On the other hand, PYCR2 overexpression significantly promoted cell viability and glycolysis, which could be inhibited by either a glycolysis inhibitor or an AKT inhibitor, indicating that PYCR2 may function via glycolysis and the AKT pathway. Moreover, despite the overexpression of PYCR2 in vivo, treatment with a glycolysis inhibitor may considerably suppress tumor growth. Our findings suggest that PYCR2 may play an oncogenic role in HCC growth by promoting glycolysis and activating AKT, emphasizing PYCR2's clinical relevance in HCC management as a novel potential therapeutic target.

Epigenetic modification facilitates proline synthase PYCR1 aberrant expression in gastric cancer.

BACKGROUND & AIMS: Pyrroline-5-carboxylate reductase 1 (PYCR1) upregulation contributes to the progression of gastric cancer (GC) and indicates poor survival. However, PYCR1 expression profile in GC subtypes and the mechanism behind its upregulation are not well-studied. METHODS: PYCR1 expression profiles in GC subtypes and different stages of gastric carcinogenesis were assessed in different GC cohorts. Genetic alterations and epigenetic modulation in PYCR1 regulation were further investigated using bioinformatics analysis and in vitro experiments. RESULTS: PYCR1 expression was significantly higher in intestinal-type GC and associated molecular subtypes in TCGA and ACRG GC cohorts. During the cascade of intestinal-type GC, PYCR1 was continuously increased from normal gastric tissues through to atrophic gastritis, to intraepithelial neoplasia, and to GC. Copy number alterations in PYCR1 were associated with PYCR1 transcript expression. One CpG island was observed in PYCR1 promoter region, and the hypomethylation occurred at this region could contribute to PYCR1 transcriptional activation in GC. Besides, H3K27ac combination was found in PYCR1 promoter, and acetyltransferase p300 induced H3K27ac could promote PYCR1 expression in GC. CONCLUSIONS: PYCR1 expression varies across GC subtypes, with intestinal-type GC and associated molecular subtypes having the highest expression. Hypomethylation at CpG sites and p300-induced H3K27ac modification within PYCR1 promoter could contribute to maintaining PYCR1 overexpression in GC. These results provide us with a new insight into epigenetic modulation in mitochondrial proline metabolism.

Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix.

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

Shikonin induces apoptosis and autophagy via downregulation of pyrroline-5-carboxylate reductase1 in hepatocellular carcinoma cells.

Shikonin(SK) is a natural small molecule naphthoquinone compound, which has anti-cancer activity in various human malignant tumors. Pyrroline-5-carboxylate reductase 1(PYCR1) is involved in tumorigenesis and regulates various cellular processes, including growth, invasion, migration, and apoptosis. However, the effect of SK and PYCR1 on apoptosis and autophagy in hepatocellular carcinoma are unclear. Our goal is to determine the internal molecular mechanism of the interaction between SK and PYCR1 and its role in the occurrence and development of liver cancer. The CCK8 assay, wound healing assay, and transwell assays show that SK and siPYCR1(gene silence PYCR1) inhibited the malignant phenotype of HCC cells, including cell viability, colony formation, migration, and invasion, respectively. The flow cytometry assays and immunofluorescence show that SK and siPYCR1 activated apoptosis and autophagy, respectively. SK induces apoptosis and autophagy in a dose-dependent manner. In addition, HCC cells were transfected with small interference fragment PYCR1 siRNA to construct siPYCR1 and SK single treatment group and co-treatment group to verify the interaction between SK and PYCR1. The Western blot identified that PI3K/Akt/mTOR signal pathway protein expression was significantly downregulated in HCC cells treated with SK and siPYCR1 together. Collectively, SK may induce apoptosis and autophagy by reducing the expression of PYCR1 and suppressing PI3K/Akt/mTOR. Thus, SK may be a promising antineoplastic drug in Hepatocellular carcinoma (HCC). SK downregulating PYCR1 might supply a theoretical foundation for the potential therapeutic application in hepatocellular carcinoma.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

The demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

PYCR in Kidney Renal Papillary Cell Carcinoma: Expression, Prognosis, Gene Regulation Network, and Regulation Targets.

BACKGROUND: Pyrroline-5-carboxylate reductase (PYCR) includes three human genes encoding three isozymes, PYCR1, PYCR2, and PYCR3 (or PYCRL), which facilitate the final step in the conversion of glutamine to proline. These genes play important roles in regulating the cell cycle and redox homeostasis as well as promoting growth signaling pathways. Proline is abnormally upregulated in a variety of cancers, and as the last key enzyme in proline production, PYCR plays an integral role in promoting tumorigenesis and cancer progression. However, its role in patients with kidney renal papillary cell carcinoma (KIRP) has not been fully elucidated. In this study, we aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of PYCR in patients with KIRP, elucidate the association between PYCR expression and KIRP, and identify potential new targets for the clinical treatment of KIRP. METHODS: We systematically analyzed the expression, prognosis, gene regulatory network, and regulatory targets of PYCR1, PYCR2, and PYCRL in KIRP using multiple online databases including cBioPortal, STRING, MethSurv, GeneMANIA, Gene Expression Profiling Interactive Analysis (GEPIA), Metascape, UALCAN, LinkedOmics, and TIMER. RESULTS: The expression levels of PYCR1, PYCR2, and PYCRL were considerably upregulated in patients with KIRP based on sample type, sex, age, and individual cancer stage. PYCR1 and PYCR2 transcript levels were markedly upregulated in females than in males, and patients aged 21-40 years had higher PYCR1 and PYCR2 transcript levels than those in other age groups. Interestingly, PYCR2 transcript levels gradually decreased with age. In addition, the expressions of PYCR1 and PYCR2 were notably correlated with the pathological stage of KIRP. Patients with KIRP with low PYCR1 and PYCR2 expression had longer survival than those with high PYCR1 and PYCR2 expression. PYCR1, PYCR2, and PYCRL were altered by 4%, 7%, and 6%, respectively, in 280 patients with KIRP. The methylation levels of cytosine-phosphate-guanine (CpG) sites in PYCR were markedly correlated with the prognosis of patients with KIRP. PYCR1, PYCR2, PYCRL, and their neighboring genes form a complex network of interactions. The molecular functions of the genes, as demonstrated by their corresponding Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, included calcium channel activity, phospholipid binding, RNA polymerase II-specificity, and kinase and GTPase-regulatory activities. PYCR1, PYCR2, and PYCRL targeted miR-21, miR-221, and miR-222, resulting in a better prognosis of KIRP. We analyzed mRNA sequencing data from 290 patients with KIRP and found that ADA, NPM3, and TKT were positively associated with PYCR1 expression; PFDN2, JTB, and HAX1 were positively correlated with PYCR2 expression; SHARPIN, YDJC, and NUBP2 were positively correlated with PYCRL expression; PYCR1 was positively correlated with B cell and CD8+ T-cell infiltration levels; macrophage infiltration was negatively correlated with PYCR2 expression; and PYCRL expression was negatively correlated with B-cell, CD8+ T cell, and dendritic cell infiltration levels. CONCLUSIONS: PYCR1, PYCR2, and PYCRL may be potential therapeutic and prognostic biomarkers for patients with KIRP. The regulation of microRNAs (miRNAs), including miR-21, miR-221, and miR-222, may prove an important strategy for KIRP treatment.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

MicroRNA hsa-miR-150-5p inhibits nasopharyngeal carcinogenesis by suppressing PYCR1 (pyrroline-5-carboxylate reductase 1).

Nasopharyngeal cancer is a rare cancer type, but with a low five-year survival rate. Dysregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) and microRNA hsa-miR-150-5p is involved in the development of various cancers. However, the molecular mechanism of the hsa-miR-150-5p-PYCR1 axis in nasopharyngeal cancer remains unclear. To identify the mechanism of the hsa-miR-150-5p-PYCR1 axis, the expression of hsa-miR-150-5p and PYCR1 in nasopharyngeal cancer tissues and cells was first measured by reverse transcription quantitative polymerase chain reaction. The luciferase and RNA pull-down assays were used to confirm the interaction between hsa-miR-150-5p and PYCR1. The overexpression of hsa-miR-150-5p and PYCR1 was detected by cell viability, proliferation, western blotting, migration, and invasion in nasopharyngeal cancer cells. The expression levels of hsa-miR-150-5p was reduced in the nasopharyngeal cancer tissues and cells and were negatively correlated with the PYCR1 levels. The upregulation of hsa-miR-150-5p significantly repressed cell growth and promoted apoptosis. However, the upregulation of PYCR1 expression significantly promoted nasopharyngeal carcinogenesis, which could abolish the inhibitory effect of hsa-miR-150-5p. In conclusion, we clarified that hsa-miR-150-5p attenuated nasopharyngeal carcinogenesis by reducing the PYCR1 expression levels. This provides a new perspective of nasopharyngeal cancer involving both hsa-miR-150-5p and PYCR1 for the treatment of nasopharyngeal cancer.

Pyrroline-5-Carboxylate Reductase-2 Promotes Colorectal Cancer Progression via Activating PI3K/AKT/mTOR Pathway.

AIM: The aim of this study was to investigate the effect and underlying pathway of pyrroline-5-carboxylate reductase-2 (PYCR2) on colorectal cancer (CRC). METHODS: The Cancer Genome Atlas (TCGA) database was used to analyze PYCR2 expression levels and clinical information. Cell proliferation was evaluated using colony forming and EdU assay. Cell apoptosis rate was determined using flow cytometry. Cell migration and invasion were measured by performing a Transwell assay, and PYCR2, MMP-2, MMP-9, Bax, cleaved caspase-3, Bcl-2, cleaved PARP, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR protein levels were detected by Western blot. RESULTS: A review of the TCGA database revealed that PYCR2 was highly expressed in CRC patients and that high PYCR2 expression was associated with advanced stage, adenocarcinoma, nodal metastasis, and poor survival rate. Moreover, PYCR2 knockdown reduced cell viability, proliferation, migration, and invasion and increased apoptosis. Additionally, PYCR2 knockdown increased Bax, cleaved caspase-3, and cleaved PARP levels and decreased Bcl-2, MMP-2, MMP-9, p-PI3K, p-AKT, and p-mTOR levels in CRC cells. Effects of silencing PYCR2 on proliferation, migration, invasion, apoptosis, and the PI3K/AKT/mTOR pathway in CRC cells were all reversed using a PI3K activator (740Y-P). CONCLUSION: PYCR2 was highly expressed in CRC, and its knockdown suppressed CRC tumorigenesis via inhibiting the activation of PI3K/AKT/mTOR pathway. This finding provides a new theoretical foundation for the treatment of CRC.

Increased PYCR1 mRNA predicts poor prognosis in kidney adenocarcinoma: A study based on TCGA database.

ABSTRACT: The pyrroline-5-carboxylate reductase 1 (PYCR1) plays important roles in cancers, but its contribution to adenocarcinoma of the kidney (AK) and the potential mechanism remain to be clarified. In this study, we aimed to demonstrate the relationship between PYCR1 mRNA and AK based on The Cancer Genome Atlas database.PYCR1 mRNA in AK and normal tissues was compared using Wilcoxon rank sum test. The relationship between PYCR1 mRNA and clinicopathological characters was evaluated using logistic regression. The association between PYCR1 mRNA and survival rate was evaluated using Kaplan-Meier test and Cox regression of univariate and multivariate analysis. Additionally, Gene Set Enrichment Analysis was conducted to annotate the biological function of PYCR1 mRNA.Increased PYCR1 mRNA was found in AK tissues. Increased PYCR1 mRNA was related to high histologic grade, clinical stage, and lymph node and distant metastasis. Kaplan-Meier survival analysis and univariate analysis showed that AK patients with increased PYCR1 mRNA had worse prognosis than those without. PYCR1 mRNA remained independently associated with overall survival (HR: 1.34; 95% CI: 1.07-1.66; P = .009) in multivariate analysis. The Gene Set Enrichment Analysis suggested that ribosome, proteasome, inhibition of p53 signaling pathway, extracellular matrix receptor interaction, and homologous recombination were differentially enriched in increased PYCR1 mRNA phenotype.Increased PYCR1 mRNA is a potential marker in patients with AK. More importantly, p53 pathway, ribosome, proteasome, extracellular matrix receptor interaction, and homologous are differentially enriched in AK patients with increased PYCR1 mRNA.

MiR-1207-5p targets PYCR1 to inhibit the progression of prostate cancer.

Prostate cancer, the most common non-cutaneous male cancer, is a public health problem with a third prevalence worldwide. PYCR1 and miR-1207-5p dysregulations were found in cancer progression. Our study aims to reveal the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression. First, we investigated the expression of miR-1207-5p in prostate cancer tissues and cell lines by RT-qPCR. Next, we confirmed miR-1207-5p targeting PYCR1 by luciferase assay. CCK-8 assay, BrdU assay, flow cytometry, and tanswell assay were applied for examining cell proliferation, apoptosis, and invasion in prostate cancer cells, respectively. In the present study, decreased miR-1207-5p expression was obviously observed in prostate cancer tissues and cells. Upregulation of miR-1207-5p hampered cellular proliferation and invasion, while enhanced cellular apoptosis. In addition, upregulation of PYCR1 elevated cell proliferation and invasion, but repressed apoptosis of prostate cancer cells. Moreover, miR-1207-5p inhibited the expression of PYCR1 to repress prostate cancer tumorigenesis. MiR-1207-5p inhibited the expression of PYCR1 to repress the progression of prostate cancer by inhibiting cell growth and elevating cell apoptosis. Overall, our study clarifies the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression, which might be effective biomarkers for clinical treatment of prostate cancer.

How signaling pathways link extracellular mechano-environment to proline biosynthesis: A hypothesis: PINCH-1 and kindlin-2 sense mechanical signals from extracellular matrix and link them to proline biosynthesis.

We propose a signaling pathway in which cell-extracellular matrix (ECM) adhesion components PINCH-1 and kindlin-2 sense mechanical signals from ECM and link them to proline biosynthesis, a vital metabolic pathway for macromolecule synthesis, redox balance, and ECM remodeling. ECM stiffening promotes PINCH-1 expression via integrin signaling, which suppresses dynamin-related protein 1 (DRP1) expression and mitochondrial fission, resulting in increased kindlin-2 translocation into mitochondria and interaction with Δ1 -pyrroline-5-carboxylate (P5C) reductase 1 (PYCR1). Kindlin-2 interaction with PYCR1 protects the latter from proteolytic degradation, leading to elevated PYCR1 level. Additionally, PINCH-1 promotes P5C synthase (P5CS) expression and P5C synthesis, which, together with increased PYCR1 level, support augmented proline biosynthesis. This signaling pathway is frequently activated in fibrosis and cancer, resulting in increased proline biosynthesis and excessive collagen matrix production, which in turn further promotes ECM stiffening. Targeting this signaling pathway, therefore, may provide an effective strategy for alleviating fibrosis and cancer progression.

Deciphering the effects of PYCR1 on cell function and its associated mechanism in hepatocellular carcinoma.

Overexpression of pyrroline-5-carboxylate reductase 1 (PYCR1) has been associated with the development of certain cancers; however, no studies have specifically examined the role of PYCR1 in hepatocellular carcinoma (HCC). Based on The Cancer Genome Atlas expression array and meta-analysis conducted using the Gene Expression Omnibus database, we determined that PYCR1 was upregulated in HCC compared to adjacent nontumor tissues (P < 0.05). These data were verified using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry analysis. Additionally, patients with low PYCR1 expression showed a higher overall survival rate than patients with high PYCR1 expression. Furthermore, PYCR1 overexpression was associated with the female sex, higher levels of alpha-fetoprotein, advanced clinical stages (III and IV), and a younger age (< 45 years old). Silencing of PYCR1 inhibited cell proliferation, invasive migration, epithelial-mesenchymal transition, and metastatic properties in HCC in vitro and in vivo. Using RNA sequencing and bioinformatics tools for data-dependent network analysis, we found binary relationships among PYCR1 and its interacting proteins in defined pathway modules. These findings indicated that PYCR1 played a multifunctional role in coordinating a variety of biological pathways involved in cell communication, cell proliferation and growth, cell migration, a mitogen-activated protein kinase cascade, ion binding, etc. The structural characteristics of key pathway components and PYCR1-interacting proteins were evaluated by molecular docking, and hotspot analysis showed that better affinities between PYCR1 and its interacting molecules were associated with the presence of arginine in the binding site. Finally, a candidate regulatory microRNA, miR-2355-5p, for PYCR1 mRNA was discovered in HCC. Overall, our study suggests that PYCR1 plays a vital role in HCC pathogenesis and may potentially serve as a molecular target for HCC treatment.

Knockdown of PYCR1 suppressed the malignant phenotype of human hepatocellular carcinoma cells via inhibiting the AKT pathway activation.

Hepatocellular carcinoma (HCC) is a common and highly malignancy tumor. Pyrroline-5-carpoxylate reductase-1 (PYCR1) is an active enzyme involved in cell metabolism. In this study, we explored the role of PYCR1 in the HCC cell lines, Hep3B and HepG2. The expression of PYCR1 was up-regulated in liver hepatocellular carcinoma (LIHC) tissue by GEPIA. Meanwhile the overall survival rate (OS) showed that patients with high PYCR1 expression had a worse prognosis compared with patients with low PYCR1 level. In addition, knockdown of PYCR1 suppressed the proliferation, invasion and migration of Hep3B and HepG2 cells and promoted the apoptosis and G1 arrest. Knockdown of PYCR1 reduced the expression of the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax and Caspase3. Furthermore, knockdown of PYCR1 changed the expression of p-AKT and its target gene Cyclin D1. In conclusion, knockdown of PYCR1 inhibited the malignant phenotype of human HCC cells by regulating the AKT pathway activation, which provides a potential strategy for the human HCC therapy.

PYCR, a key enzyme in proline metabolism, functions in tumorigenesis.

Pyrroline-5-carboxylate reductase (PYCR), the last enzyme in proline synthesis that converts P5C into proline, was found promoting cancer growth and inhibiting apoptosis through multiple approaches, including regulating cell cycle and redox homeostasis, and promoting growth signaling pathways. Proline is abnormally up-regulated in multiple cancers and becomes one of the critical players in the reprogramming of cancer metabolism. As the last key enzymes in proline generation, PYCRs have been the subject of many investigations, and have been demonstrated to play an indispensable role in promoting tumorigenesis and cancer progression. In this article, we will thoroughly review the recent investigations on PYCRs in cancer development.

Isozymes of P5C reductase (PYCR) in human diseases: focus on cancer.

Δ1-Pyrroline-5-carboxylate (P5C) reductase (PYCR or P5CR) catalyzes the conversion of P5C to L-proline (Pro) with concomitant oxidation of a cofactor, NADPH or NADH. Mammalian PYCR have been studied since 1950' and currently three isozymes of human PYCR, 1, 2, and L, have been identified and characterized and their roles in genetic diseases and cancer biology have been keenly investigated. These three isozymes are encoded by three different genes localized at three different chromosomes, and catalyze NAD(P)H-dependent reduction of P5C to Pro important for the transfer of oxidizing potential across the mitochondrion and cell. The review summarizes the current understanding of these three human PYCR isozymes and their roles in diseases with a focus on cancer.

The upregulation of PYCR2 is associated with aggressive colon cancer progression and a poor prognosis.

PYCR2 has previously been shown to be related to a range of malignancies including hepatocellular carcinoma and melanoma, but its mechanistic functions and prognostic relevance in colon cancer patients remain to be defined. Herein, we used the Oncomine, Human Protein Atlas, The Cancer Genome Atlas (TCGA), and UALCAN databases to explore the expression of this gene in different human cancer, after which the relationship between PYCR2 expression and patient clinicopathologic characteristics was evaluated. We utilized an in vitro approach to evaluate the association between PYCR2 expression and colon cancer cell proliferation, migration, invasion, and tumor microsphere formation. The cell apoptosis was analyzed by flow cytometry. Gene set enrichment analysis (GSEA) approaches were additionally used to probe signaling pathways related to PYCR2. These analyses confirmed that PYCR2 was upregulated in several cancer types including colon cancer, with such upregulation correlating with a poor patient prognosis and with malignant clinicopathological characteristics. PYCR2 expression was identified as an independent predictor of colon cancer patients' survival, and in vitro analyses suggested that knocking down this gene was sufficient to disrupt the proliferative, migratory, invasive, and microsphere formation activities of colon cancer cells. Moreover, shPYCR2 transfection induced colon cancer cell apoptosis. GSEA suggested that high PYCR2 expression correlates with the differential enrichment of the Wnt ß-catenin signaling, MYC targets, RNA polymerase, and Notch signaling pathways. Overall, these data indicate that PYCR2 is an important mediator of tumor progression and metastasis, and suggest that it may be a valuable prognostic indicator for colon cancer patient evaluation.

Genetic analysis of Pycr1 and Pycr2 in mice.

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.