Thymidine Phosphorylase Expression Research Articles

Analyzing the Impact of Oncological Data at Different Time Points and Tumor Biomarkers on Artificial Intelligence Predictions for Five-Year Survival in Esophageal Cancer

AIM: In this study, we use Artificial Intelligence (AI), including Machine (ML) and Deep Learning (DL), to predict the long-term survival of resectable esophageal cancer (EC) patients in a high-volume surgical center. Our objective is to evaluate the predictive efficacy of AI methods for survival prognosis across different time points of oncological treatment. This involves comparing models trained with clinical data, integrating either Tumor, Node, Metastasis (TNM) classification or tumor biomarker analysis, for long-term survival predictions. METHODS: In this retrospective study, 1002 patients diagnosed with EC between 1996 and 2021 were analyzed. The original dataset comprised 55 pre- and postoperative patient characteristics and 55 immunohistochemically evaluated biomarkers following surgical intervention. To predict the five-year survival status, four AI methods (Random Forest RF, XG Boost XG, Artificial Neural Network ANN, TabNet TN) and Logistic Regression (LR) were employed. The models were trained using three predefined subsets of the training dataset as follows: (I) the baseline dataset (BL) consisting of pre-, intra-, and postoperative data, including the TNM but excluding tumor biomarkers, (II) clinical data accessible at the time of the initial diagnostic workup (primary staging dataset, PS), and (III) the PS dataset including tumor biomarkers from tissue microarrays (PS + biomarkers), excluding TNM status. We used permutation feature importance for feature selection to identify only important variables for AI-driven reduced datasets and subsequent model retraining. RESULTS: Model training on the BL dataset demonstrated similar predictive performances for all models (Accuracy, ACC: 0.73/0.74/0.76/0.75/0.73; AUC: 0.78/0.82/0.83/0.80/0.79 RF/XG/ANN/TN/LR, respectively). The predictive performance and generalizability declined when the models were trained with the PS dataset. Surprisingly, the inclusion of biomarkers in the PS dataset for model training led to improved predictions (PS dataset vs. PS dataset + biomarkers; ACC: 0.70 vs. 0.77/0.73 vs. 0.79/0.71 vs. 0.75/0.69 vs. 0.72/0.63 vs. 0.66; AUC: 0.77 vs. 0.83/0.80 vs. 0.85/0.76 vs. 0.86/0.70 vs. 0.76/0.70 vs. 0.69 RF/XG/ANN/TN/LR, respectively). The AI models outperformed LR when trained with the PS datasets. The important features shared after AI-driven feature selection in all models trained with the BL dataset included histopathological lymph node status (pN), histopathological tumor size (pT), clinical tumor size (cT), age at the time of surgery, and postoperative tracheostomy. Following training with the PS dataset with biomarkers, the important predictive features included patient age at the time of surgery, TP-53 gene mutation, Mesothelin expression, thymidine phosphorylase (TYMP) expression, NANOG homebox protein expression, and indoleamine 2,3-dioxygenase (IDO) expressed on tumor-infiltrating lymphocytes, as well as tumor-infiltrating Mast- and Natural killer cells. CONCLUSION: Different AI methods similarly predict the long-term survival status of patients with EC and outperform LR, the state-of-the-art classification model. Survival status can be predicted with similar predictive performance with patient data at an early stage of treatment when utilizing additional biomarker analysis. This suggests that individual survival predictions can be made early in cancer treatment by utilizing biomarkers, reducing the necessity for the pathological TNM status post-surgery. This study identifies important features for survival predictions that vary depending on the timing of oncological treatment.

A pilot study investigating plasma pharmacokinetics and tolerance of oral capecitabine in carcinoma-bearing dogs

BackgroundCapecitabine is an oral prodrug of the active metabolite 5-fluorouracil, which has been used effectively in human colorectal, head and neck, and mammary carcinomas. Capecitabine has several properties that make it an attractive treatment option for dogs: (i) it is relatively inexpensive, (ii) it has a short half-life in humans, allowing for rapid plasma concentration changes to be achieved with dosage adjustments, (iii) it is effective for treating carcinomas in humans, for which there are no widely-effective oral chemotherapy options in dogs, and (iv) it is thought to preferentially target cancer cells due to different expression of thymidine phosphorylase, thereby decreasing the risk of off-target side effects. However, capecitabine has not been widely explored as a chemotherapy agent for dogs. The goal of this study was to determine the plasma disposition of capecitabine in dogs following a single oral dose and to document any adverse events associated with capecitabine administration over the course of 5 weeks.ResultsCapecitabine was well tolerated throughout the 5-week study period when administered to 5 dogs with naturally occurring carcinomas at 750 mg/m2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document} by mouth once daily for 14 consecutive days in a 3-week cycle. No dogs withdrew from the study due to adverse events or other causes. The median AUC0-last\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{\ ext {0-last}}$$\\end{document} was 890 h·\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\cdot$$\\end{document}ng/ml (range 750-1100 h·\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\cdot$$\\end{document}ng/ml); however, the maximum blood concentration and time to reach that concentration of capecitabine was highly variable after a single dose.ConclusionsCapecitabine appears well-tolerated as an oral chemotherapy agent for dogs with carcinomas, although individualized dosing may be necessary, and further studies are warranted.

Abstract 12759: Thymidine Phosphorylase Enhances SARS-CoV-2 Spike Protein and Platelet Factor 4 Complex Formation: A Potential Novel Mechanism That May Account for COVID-19-Associated Thrombosis

Introduction: Scattered studies suggest that SARS-CoV-2 spike protein (SP) promotes platelet activation, though the exact mechanism is unknown. Thymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in the plasma and lungs of COVID-19 patients, suggesting that TYMP may play a role in the COVID-19 milieu. Hypothesis: We hypothesize that TYMP enhances SP/platelet factor 4 (PF4) complex formation and inhibition of TYMP attenuates SP-induced thrombosis and/or inflammation. Methods: BEAS-2B cells were treated with SP- or its receptor binding domain (RBD)-containing COS-7 cell lysate, or control cell lysate (p3.1) and TYMP and STAT3 expression were determined by western blot. The effect of SP on thrombosis was examined in K18-hACE2 and K18-hACE2/ Tymp -/- mice using the ferric chloride-induced carotid artery injury thrombosis model. SP-, TYMP-, or PF4-encoding plasmids were co-transfected into COS-7 and their protein interactions were assessed using co-IP, Blue Native-PAGE, and immunocytochemistry. Results: SP increased expression of TYMP and pY705-STAT3 in BEAS-2B. siRNA-mediated knockdown of TYMP reduced STAT3 activation. SP significantly enhanced thrombosis in the K18-hACE2 mice, which was inhibited by simultaneously treating mice with tipiracil, a selective TYMP inhibitor ( Figure ). K18-hACE2/ Tymp -/- mice were resistant to SP-enhanced thrombosis. For the first time, we found that SP, PF4, and TYMP form a complex whose formation is dose-dependently enhanced by TYMP. Conclusions: Our studies suggest that SARS-CoV-2 SP enhances TYMP expression and is prothrombotic and proinflammatory. TYMP inhibition, either via genetic knockout or by its chemical inhibitor, attenuates SP-enhanced thrombosis. TYMP promotes the formation of a PF4/SP complex, which provides a novel insight into COVID-19-associated thrombosis. TYMP could be a novel therapeutic target for COVID-19-associated sequelae.

Rs11479 in Thymidine Phosphorylase Associated with Prognosis of Patients with Colorectal Cancer Who Received Capecitabine-Based Adjuvant Chemotherapy.

Thymidine Phosphorylase (TYMP) gene was of potential significance in the process of colorectal cancer (CRC) development and played an important role in capecitabine metabolism. This study was to identify the association between TYMP polymorphism and prognosis of postoperative patients with CRC who received capecitabine-based adjuvant chemotherapy. A total of 218 patients with CRC who were treated with surgical resection and capecitabine-based adjuvant chemotherapy were included in this study retrospectively. Peripheral blood and peripheral blood mononuclear cell (PBMC) specimen of the patients were collected for the genotyping of TYMP polymorphism and TYMP mRNA expression, respectively. Univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, Cox regression analysis was adopted in multivariate analysis. The mRNA expression of TYMP according to genotype status was analyzed using non-parameter test. Prevalence of rs11479 in TYMP among the 218 patients exhibited that minor allele frequency of rs11479 was 0.20 (GG 141 cases, GA 68 cases and AA 9 cases), which was in accordance with Hardy-Weinberg equilibrium (P=0.825). Association analysis suggested that the median disease-free survival (DFS) of patients with GG genotype and GA/AA genotype was 3.1 and 6.1 years, respectively (P=0.004). Furthermore, the median overall survival of patients with GG genotype and GA/AA genotype was 5.0 and 7.0 years, respectively (P=0.033). Multivariate Cox regression analysis exhibited that rs11479 polymorphism was an independent factor for DFS (HR = 1.64, P=0.009). Additionally, of the 65 PBMC specimens, mRNA expression results indicated that patients with GA/AA genotypes conferred significantly higher mRNA expression of TYMP than that of patients with GG genotype (P<0.001). Polymorphism rs11479 in TYMP gene might predict the prognosis of patients with CRC who received capecitabine-based adjuvant chemotherapy through mediation of the mRNA expression of TYMP. The conclusion of this study should be validated in prospective clinical trials subsequently.

Depletion of plasma thymidine results in growth retardation and mitochondrial myopathy in mice overexpressing human thymidine phosphorylase

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.

Effects of gene expression in 5-FU metabolic pathways in a phase III trial evaluating adjuvant S-1 therapy compared to surgery alone following curative resection for biliary tract cancer (JCOG1202A1).

732 Background: S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy in Japanese patients with resected biliary tract cancer (BTC), based on the results of JCOG1202, a phase III trial evaluating adjuvant S-1 following curative resection for BTC compared to surgery alone. The efficacy of 5-FU-based therapy is related to the expression of enzymes involved in 5-FU metabolic pathways, such as thymidylate synthase (TS), thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD). The aim of this study was to evaluate the impact of messenger RNA (mRNA) levels of the four 5-FU metabolic pathway genes on the outcomes of patients enrolled in JCOG1202. Methods: Tumor cell RNA was isolated from formalin-fixed paraffin-embedded primary BTC specimens resected in 264 of 440 randomized patients in JCOG1202. The four 5-FU metabolic pathway genes were measured in 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89) who were randomly divided to training (n = 96) or validation set (n = 87). The endpoints of interest were the predictive values of the four genes for the efficacy of adjuvant S-1 on overall survival and relapse-free survival (RFS), and we here report the results regarding RFS. Cut-off levels for mRNA expression were selected in the training set which minimized the bootstrap p-values (2,000 samples) of an interaction term of treatment (surgery alone or S-1) and mRNA expression in a Cox regression model. Results: There were no obvious differences in each mRNA level and clinical characteristics between surgery alone and adjuvant S-1 groups. RFS tended to be better with adjuvant S-1 (hazard ratio [HR] = 0.790, [95% confidence interval: 0.524-1.192]) compared to surgery alone, which was maintained in the low DPD population (HR = 0.440 [0.216-0.898] in training set and 0.748 [0.334-1.675] in validation set), in the low TP population (HR = 0.709 [0.388-1.296] and 0.602 [0.287-1.262]), and in the high OPRT population (HR = 0.520 [0.152-1.779] and 0.609 [0.161-2.304]). Conclusions: The efficacy of adjuvant S-1 on RFS was representative in BTC patients with intratumoral gene expressions of low DPD, low TP, and high OPRT, which was disappeared in the population with high DPD, high TP, and low OPRT. These biomarkers might be useful to predict therapeutic benefits with S-1 containing chemotherapy for BTC.

Identifying TYMP as an Immune Prognostic Marker in Clear Cell Renal Cell Carcinoma.

In clear cell renal cell carcinoma (ccRCC), only some patients can benefit from immunotherapy therapy, and it is urgent to find immune-related molecular markers and targets. Thymidine phosphorylase (TYMP) expression level and predictive value in pan-cancers were analyzed using TIMER, GEPIA2, and The Human Protein Atlas. We obtained ccRCC tissues to verify the differential expression of TYMP and confirmed the biological function in vitro. Subsequently, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) are used to explore the potential mechanism of TYMP. Finally, TIMER was used to analyze the infiltration levels and prognostic value of different immune cells. TYMP is upregulated in various cancers, including ccRCC, and there is a certain degree of causality between high expression and poor prognosis in ccRCC. It was confirmed that TYMP knockdown could suppress cell aggressiveness, and cause cell death. Differential analysis showed that 55 differential genes were upregulated in the high-expression groups of TYMP. KEGG and GSEA analyses suggested that TYMP was linked to immune cell invasion, fatty acid metabolism, and P53 signaling pathway. Further investigation revealed that the expression level of TYMP linked positively to T-cell follicular helper and Tregs, but negatively with mast cell activation. Finally, a Nomogram was established on the base of expression level of TYMP and the clinical characteristics of ccRCC patients to predict prognosis. Patient survival is poor and immune cell infiltration is abnormal when TYMP is highly expressed in ccRCC, suggesting that ccRCC patients could benefit from using TYMP as a molecular diagnostic and therapeutic target.

Capecitabine induces hand-foot syndrome through elevated thymidine phosphorylase-mediated locoregional toxicity and GSDME-driven pyroptosis that can be relieved by tipiracil.

Hand-foot syndrome (HFS) is a serious dose-limiting cutaneous toxicity of capecitabine-containing chemotherapy, leading to a deteriorated quality of life and negative impacts on chemotherapy treatment. The symptoms of HFS have been widely reported, but the precise molecular and cellular mechanisms remain unknown. The metabolic enzyme of capecitabine, thymidine phosphorylase (TP) may be related to HFS. Here, we investigated whether TP contributes to the HFS and the molecular basis of cellular toxicity of capecitabine. TP-/- mice were generated to assess the relevance of TP and HFS. Cellular toxicity and signalling mechanisms were assessed by in vitro and in vivo experiments. TP-/- significantly reduced capecitabine-induced HFS, indicating that the activity of TP plays a critical role in the development of HFS. Further investigations into the cellular mechanisms revealed that the cytotoxicity of the active metabolite of capecitabine, 5-DFUR, was attributed to the cleavage of GSDME-mediated pyroptosis. Finally, we demonstrated that capecitabine-induced HFS could be reversed by local application of the TP inhibitor tipiracil. Our findings reveal that the presence of elevated TP expression in the palm and sole aggravates local cell cytotoxicity, further explaining the molecular basis underlying 5-DFUR-induced cellular toxicity and providing a promising approach to the therapeutic management of HFS.

Combination of T cell-redirecting bispecific antibody ERY974 and chemotherapy reciprocally enhances efficacy against non-inflamed tumours

Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.

Ginseng-derived nanoparticles inhibit lung cancer cell epithelial mesenchymal transition by repressing pentose phosphate pathway activity.

It is unclear whether ginseng-derived nanoparticles (GDNPs) can prevent tumor cell epithelial-mesenchymal transition (EMT). Here, we describe typical characteristics of GDNPs and possible underlying mechanisms for GDNP antitumor activities. First, GDNPs particle sizes and morphology were determined using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively, while cellular uptake of PKH67-labeled GDNPs was also assessed. Next, we evaluated GDNPs antitumor effects by determining whether GDNPs inhibited proliferation and migration of five tumor cell lines derived from different cell types. The results indicated that GDNPs most significantly inhibited proliferation and migration of lung cancer-derived tumor cells (A549, NCI-H1299). Moreover, GDNPs treatment also inhibited cell migration, invasion, clonal formation, and adhesion tube formation ability and reduced expression of EMT-related markers in A549 and NCI-H1299 cells in a dose-dependent manner. Meanwhile, Kaplan-Meier analysis of microarray data revealed that high-level thymidine phosphorylase (TP) production, which is associated with poor lung cancer prognosis, was inhibited by GDNPs treatment, as reflected by decreased secretion of overexpressed TP and downregulation of TP mRNA-level expression. In addition, proteomic analysis results indicated that GDNPs affected pentose phosphate pathway (PPP) activity, with ELISA results confirming that GDNPs significantly reduced levels of PPP metabolic intermediates. Results of this study also demonstrated that GDNPs-induced downregulation of TP expression led to PPP pathway inhibition and repression of lung cancer cell metastasis, warranting further studies of nano-drugs as a new and promising class of anti-cancer drugs.

Heat shock protein 90 inhibitor 17-AAG down-regulates thymidine phosphorylase expression and potentiates the cytotoxic effect of tamoxifen and erlotinib in human lung squamous carcinoma cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, in cancer cells, are related to a poor prognosis in a variety of cancers. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is involved in the stabilization and maturation of many oncogenic proteins. The aim of this study is to elucidate whether Hsp90 inhibitor 17-AAG could enhance tamoxifen- and erlotinib-induced cytotoxicity in nonsmall cell lung cancer (NSCLC) cells via modulating TP expression in two squamous NSCLC cell lines, H520 and H1703. We found that 17-AAG reduced TP expression via inactivating the MKK1/2-ERK1/2-mitogen-activated protein kinase (MAPK) pathway. TP knockdown with siRNA or ERK1/2 MAPK inactivation with the pharmacological inhibitor U0126 could enhance the cytotoxic and growth inhibitory effects of 17-AAG. In contrast, MKK1-CA or MKK2-CA (a constitutively active form of MKK1/2) vector-enforced expression could reduce the cytotoxic and cell growth inhibitory effects of 17-AAG. Furthermore, 17-AAG enhanced the cytotoxic and cell growth inhibitory effects of tamoxifen and erlotinib in NSCLC cells, which were associated with TP expression downregulation and MKK1/2-ERK1/2 signal inactivation. Taken together, Hsp90 inhibition downregulates TP, enhancing the tamoxifen- and erlotinib-induced cytotoxicity in H520 and H1703 cells.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer

BackgroundTumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance.MethodsStage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma.ResultsTYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p < 0.05).ConclusionsTYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Abstract 130: Thymidine Phosphorylase Enhances Abdominal Aortic Aneurysm Formation In Mice

Abdominal aortic aneurysm (AAA) is affected by multiple factors, including vessel wall cells, extracellular matrix, genetic disorders, etc.; however, the pathophysiology of AAA remains unclear. We recently found that thymidine phosphorylase (TYMP) is a signaling protein, and an increase of TYMP inhibits the proliferation of VSMCs, the major cell population that affects AAA progression. TYMP enhances platelet activity, thrombosis, and inflammation. These data suggest that TYMP may play a role in AAA formation. By using human AAA samples, we found that TYMP expression was significantly increased in AAA vessel walls than in healthy aortas. We thus further examined the role of TYMP in AAA formation using a mouse model. WT C57BL/6J and Tymp -/- mice were fed a western diet (TD.88137), started at 4 weeks (wks) old for 8 wks, and then received implantation of Ang II mini pump, which delivers Ang II to mice at a dose of 1 μg/kg/min for 4 wks. By echography, we found that Ang II perfusion dramatically increased inner aorta diameter at the diastolic phase in WT, but not Tymp -/- mice. Mice were sacrificed 4 wks after Ang II perfusion. AAA was confirmed in 3 of the 12 WT mice, but no AAA was found in all 11 Tymp -/- mice used. The AAA was mainly formed at the suprarenal artery level. Histological examination confirmed the presence of freshly formed or fibrotic hematoma. By using TYMP overexpressing rat VSMC cell line, C2, and its control cells, PC, we found that TYMP increased MMP2 production, secretion, and activation, which was diminished by tipiracil, a selective TYMP inhibitor. Ang II did not affect extracellular MMP2 activity in both PC and C2. TYMP facilitated TNF-α induced MMP2 activation. TYMP induced constitutive AKT phosphorylation, which was further enhanced by TNF-α. TYMP increased expression of active TGFβ1. Consequently, TYMP significantly enhanced thrombospondin-1 type 1 repeat domain (TSR) stimulated TGFβ1 signaling activation and expression of CTGF, which is correlated with severe fibrotic disorders and AAA. In summary, our study, for the first time, demonstrated that TYMP, a pyrimidine salvage pathway enzyme, plays an important role in regulating vascular biology, especially, VSMC function. TYMP-mediated microenvironment changes enhance the development of AAA.

Effect of Thymidine Phosphorylase Gene Demethylation on Sensitivity to 5-Fluorouracil in Colorectal Cancer Cells.

Chemotherapy is used for recurrent and metastatic colorectal cancer, but the response rate of 5-fluorouracil (5-FU), the standard treatment for colorectal cancer, is low. We hypothesized that thymidine phosphorylase (TYMP) expression, a rate-limiting activating enzyme of 5-FU, is regulated by methylation of the gene promoter region, and demethylation of TYMP would increase sensitivity to 5-FU. HCT116 colon cancer cells were treated with 5-aza-2'-deoxycytidine, a demethylating agent, and changes in TYMP transcription and sensitivity to 5-FU were evaluated. TYMP expression increased over 54-fold in HCT116 transfected with TYMP. The cytotoxicity of 5-FU increased up to 5.5-fold. In comparison, in HCT116 treated with 5-aza-2'-deoxycytidine, TYMP expression increased 5.8-fold. However, the cytotoxicity of 5-FU remained unchanged. Demethylating agent alone did not promote the cytotoxicity of 5-FU against colorectal cancer. To further increase the sensitivity to 5-FU, combination with adjuvant therapy focusing on metabolic pathways other than the TYMP pathway appear necessary.

Thymidine phosphorylase induction by ionizing radiation antagonizes 5-fluorouracil resistance in human ductal pancreatic adenocarcinoma

Chemoresistance in pancreatic ductal adenocarcinoma (PDAC) frequently contributes to failure of systemic therapy. While the radiosensitizing properties of 5-fluorouracil (FU) are well known, it is unknown whether ionizing radiation (IR) sensitizes towards FU cytotoxicity. Here, we hypothesize that upregulation of thymidine phosphorylase (TP) by IR reverses FU chemoresistance in PDAC cells. The FU resistant variant of the human PDAC cell line AsPC-1 (FU-R) was used to determine the sensitizing effects of IR. Proliferation rates of FU sensitive parental (FU-S) and FU-R cells were determined by WST-1 assays after low (0.05 Gy) and intermediate dose (2.0 Gy) IR followed by FU treatment. TP protein expression in PDAC cells before and after IR was assessed by Western blot. To analyze the specificity of the FU sensitizing effect, TP was ablated by siRNA. FU-R cells showed a 2.7-fold increase of the half maximal inhibitory concentration, compared to FU-S parental cells. Further, FU-R cells showed a concomitant IR resistance towards both doses applied. When challenging both cell lines with FU after IR, FU-R cells had lower proliferation rates than FU-S cells, suggesting a reversal of chemoresistance by IR. This FU sensitizing effect was abolished when TP was blocked by anti-TP siRNA before IR. An increase of TP protein expression was seen after both IR doses. Our results suggest a TP dependent reversal of FU-chemoresistance in PDAC cells that is triggered by IR. Thus, induction of TP expression by low dose IR may be a therapeutic approach to potentially overcome FU chemoresistance in PDAC.

Downregulation of Thymidine Phosphorylase Expression by 17-AAG Potentiates the Cytotoxic Effect of Tamoxifen and Erlotinib in Human Squamous Cell Carcinoma Cells

Downregulation of Thymidine Phosphorylase Expression by 17-AAG Potentiates the Cytotoxic Effect of Tamoxifen and Erlotinib in Human Squamous Cell Carcinoma Cells

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase.

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

STING-driven interferon signaling triggers metabolic alterations in pancreas cancer cells visualized by [18F]FLT PET imaging

Type I interferons (IFNs) are critical effectors of emerging cancer immunotherapies designed to activate pattern recognition receptors (PRRs). A challenge in the clinical translation of these agents is the lack of noninvasive pharmacodynamic biomarkers that indicate increased intratumoral IFN signaling following PRR activation. Positron emission tomography (PET) imaging enables the visualization of tissue metabolic activity, but whether IFN signaling-induced alterations in tumor cell metabolism can be detected using PET has not been investigated. We found that IFN signaling augments pancreatic ductal adenocarcinoma (PDAC) cell nucleotide metabolism via transcriptional induction of metabolism-associated genes including thymidine phosphorylase (TYMP). TYMP catalyzes the first step in the catabolism of thymidine, which competitively inhibits intratumoral accumulation of the nucleoside analog PET probe 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT). Accordingly, IFN treatment up-regulates cancer cell [18F]FLT uptake in the presence of thymidine, and this effect is dependent upon TYMP expression. In vivo, genetic activation of stimulator of interferon genes (STING), a PRR highly expressed in PDAC, enhances the [18F]FLT avidity of xenograft tumors. Additionally, small molecule STING agonists trigger IFN signaling-dependent TYMP expression in PDAC cells and increase tumor [18F]FLT uptake in vivo following systemic treatment. These findings indicate that [18F]FLT accumulation in tumors is sensitive to IFN signaling and that [18F]FLT PET may serve as a pharmacodynamic biomarker for STING agonist-based therapies in PDAC and possibly other malignancies characterized by elevated STING expression.

Evaluation of Thymidine Phosphorylase Inhibitors in Glioblastoma and Their Capacity for Temozolomide Potentiation.

A number of studies have shown high levels of thymidine phosphorylase (TP) expression in glioblastoma (GBM), with trace or undetectable TP levels in normal developed brain tissue. TP catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, maintaining nucleoside homeostasis for efficient DNA replication and cell division. The TP-mediated catabolism of thymidine is responsible for multiple protumor processes and can support angiogenesis, glycation of proteins, and alternative metabolism. In this study, we examined the effect of TP inhibition in GBM using the known nanomolar TP inhibitors 5-chloro-6-[1-(2'-iminopyrrolidin-1'-yl)methyl]uracil (TPI) and the analogous 6-[(2'-aminoimidazol-1'-yl)methyl]uracils. Although these TP inhibitors did not demonstrate any appreciable cytotoxicity in GBM cell lines as single agents, they did enhance the cytotoxicity of temozolomide (TMZ). This pontetiated action of TMZ by TP inhibition may be due to limiting the availability of thymine for DNA repair and replication. These studies support that TP inhibitors could be used as chemosensitizing agents in GBM to improve the efficacy of TMZ.

1211-P: The Mechanistic Role of Thymidine Phosphorylase in the Development of Obesity

Obesity is a major independent risk factor for the development of type II diabetes (T2DM), nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD). Consequently, controlling obesity constitutes a key strategy for reducing the prevalence of these diseases. The majority of drugs developed for treating obesity over the past 20 years have been withdrawn due to the increased risk of cardiovascular and psychiatric complications. Therefore, developing novel mechanism-mediated therapeutics for controlling obesity and its consequent complications is a top priority and urgently needed. Our studies indicate that thymidine phosphorylase (TYMP) could be a promising target. The traditional role of TYMP is to catalyze the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose-1 phosphate and maintain the homeostatic nuclear pool of these molecules. TYMP expression is increased in several diseased conditions, including obesity, T2DM, and NAFLD. However, whether TYMP plays a role in the development of these diseases is unknown. We found that Tymp-deficient (Tymp-/-) male mice exhibited significantly limited body weight gain as well as liver and perigonadal fat weight when fed either a western diet (TD.88137) or a prodiabetic high-fat diet (D12492) compared with the wild-type mice. Male Tymp-/- mice showed better glucose tolerance. TYMP-deficiency reduced expression of enzymes that confer glycolysis and de novo lipogenesis in the liver. TYMP-deficiency enhanced the sensitivity of primary hepatocytes to insulin as evidenced by the increase of AKT activation. Inhibition of TYMP with tipiracil, a selective, potent, and the FDA approved TYMP inhibitor, dramatically reduced western diet and high-fat diet-induced body weight gain. We conclude that TYMP plays a pivotal role in liver glucose and lipid metabolism, which promotes the development of obesity and T2DM. Targeting TYMP with tipiracil could be a new therapy for obesity. Disclosure H. Yue: None. A. M. Belcher: None. A. Bicak: None. W. Li: None.