Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients’ prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.

Immunohistochemical expression of thymidylate synthase and prognosis in gastric cancer patients submitted to fluoropyrimidine-based chemotherapy

Objective： Adjuvant chemotherapy with 5-fluorouracil （5-FU） has been widely used in gastric cancer （GC） patients to prevent relapse after curative resection. 5-FU acts by inhibiting thymidylate synthase （TS）, and high levels of TS correlate with resistance to treatment with fluoropyfimidines. The aim of this study was to evaluate the expression of TS in GC patients, and its relation with clinicopathological characteristics and prognosis in adjuvant chemotherapy with 5-FU. Methods： We retrospectively evaluated 285 patients who underwent D2-gastrectomy with curative intent. TS expression was determined by immunohistochemistry （IHC） in tumor cells by tissue microarray （TMA）. TS level was evaluated according to the intensity and percentage of cells marked by a score system. Patients were divided in three groups according to their TS-score： negative, low and high. Results： TS expression was positive in 92.3% of GC. TS-high, TS-low and TS-negative were observed in 46.3%, 46.0% and 7.7% of patients, respectively. High-TS GC were associated with older age （P=0.007）, high neutrophil/lymphocyte ratio （P=0.048）, well/moderately differentiated histology （P=0.001）, intestinal Lauren type （P〈0.001） and absence of perineural invasion （P=0.003）. Among 285 patients, 133 stage IUIII patients （46.7%） received chemotherapy with 5-FU. In survival analysis, TS-high was associated with worse disease-free survival （DFS） in stage III GC patients who received 5-FU-based chemotherapy （P=0.007）. Multivariate analysis revealed that total gastrectomy, poorly differentiated tumors and high TS-score were associated with worse DFS in stage III GC patients. Conclusions： High TS-score in stage III GC was associated with poor DFS in patients treated with fluoropyrimidine-based chemotherapy.

Structural analysis of a shrimp thymidylate synthase reveals species-specific interactions with dUMP and raltitrexed

Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical value,TS is supposed to be a promising drug target in aquatic-disease control.To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control,we report the crystal structures of TS from Litopenaeus vannamei(LvTS)in the apo form,LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27Å,1.54Å,and 1.56Åresolution,respectively.LvTS shares a similar fold with known TSs,existing as a dimer in the crystal.The apo LvTS and LvTS-dUMP take an open conformation,and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed.Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation,the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed;the distance between C6 of dUMP and Sγof the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations,resembling that in the TS structures with open conformations.Other species-specific interactions with dUMP and raltitrexed are also observed.Therefore,LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs.Our study provides the first crustcean TS structure,and reveals species-specific interactions between TSs and the ligands,which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.

Thymidylate synthase confers pemetrexed resistance of non-small cell lung cancer cells by EGFR/PI3K/AKT pathway

Chemotherapy drug resistance is the main cause leading to the relapse and metastasis of non-small cell lung cancer(NSCLC)patients.Our study aimed to investigate the mechanism of pemetrexed resistance in NSCLC.Firstly,the pemetrexed(PEM)-resistant PC-9 and A549 lung adenocarcinoma cell lines(PC-9/PEM and A549/PEM)were established.The expression of thymidylate synthase(TS)in PC-9/PEM,A549/PEM,A549,and PC-9 cells were analyzed by qRT-PCR and western blot.Then,cell viability,colony formation,migration,and invasion were performed on PEM-resistant cells transfected with TS siRNA.The role of EGFR in PEM resistance of PEM-resistant cells was investigated using EGFR siRNA.The effects of gefitinib and EGFR siRNA on EGFR/PI3K/AKT pathway and downstream signaling Cyclin D1 and E2F1 in PEM-resistant cells were analyzed.Results showed that the protein level of TS was significantly increased in A549/PEM and PC-9/PEM.TS knockdown inhibited the potency of proliferation,colony-forming potential,migration,and invasion in PEM-resistant cells.EGFR knockdown abrogated the resistance to PEM of PEM-resistant cells and suppressed the migration and invasion of PEM-resistant cells.Gefitinib treatment and EGFR knockdown respectively inhibited the EGFR/PI3K/AKT pathway and downregulated Cyclin D1 and E2F1 in PEM-resistant cells.Thus,TS might be a predictive marker for PEM resistance in NSCLC.Inhibition of the EGFR pathway abrogated the resistance to PEM and inhibited the EGFR/PI3K/AKT and downstream signaling of PEM-resistant NSCLC cell lines.

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM.OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models.METHODS Cell growth assays,cell cycle analysis,immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s)of action of the formulason human CRC cells.In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with 5-FU.RESULTS We identified Huangqin Gegen Tang(HQGGT)which suppressed the in vivo growth of human CRC HT-29 xenografts.HQGGT significantly inhibited the growth of CRC cell lines.HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells(H630R1)and mouse colon cancer cells(MC38).This synergy was the result of suppression of thymidylate synthase expression by HQGGT.HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts.Ongoing studies have identified Huangqin as the herb responsible for TS inhibi⁃tion.CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrim⁃idine chemotherapy for CRC treatment.

Associations between gene polymorphisms of thymidylate synthase with its protein expression and chemosensitivity to 5-fluorouracil in pancreatic carcinoma cells

Background Thymidylate synthase （TS） is a key regulatory enzyme for de novo DNA synthesis.TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs,and its expression may be affected by gene polymorphisms.In this study,we investigated the associations between polymorphisms of the TS gene and its protein expression,and the implications on the efficacy of 5-fluorouracil （5-FU） in pancreatic cancer cells.Methods Genotypes based on the 28-bp TS tandem repeat for pancreatic cell lines were determined by electrophoretic analysis of PCR products.A single nucleotide polymorphism （SNP） at nucleotide 12 of the second 28-bp repeat of the 3R allele was determined by nucleotide sequencing.The chemosensitivity of pancreatic carcinoma cells to 5-FU in vitro was evaluated using Cell Counting Kit-8 （CCK-8）.TS protein expression was analyzed by Western blotting.Results Seven pancreatic carcinoma cell lines had different genotypes in terms of the 28-bp TS tandem repeat,as follows：homozygous 2R/2R （T3M4 and BxPC-3 cells）,heterozygous 2R/3R （AsPC-1,Capan-1,and SU86.86）,and homozygous 3R/3R （PANC-1 and COLO357）.The optical density ratio of genotypes 3R/3R,2R/2R and 2R/3R was 1.393±0.374,0.568±0.032 and 0.561±0.056,respectively.Cells with the 2R/3R or 3R/3R genotypes were further analyzed for the G to C SNP at nucleotide 12 of the second 28-bp repeat of the 3R allele,yielding heterozygous 2R/3Rc （AsPC-1,Capan-1,and SU86.86）,homozygous 3Rg/3Rg （COLO357） and homozygous 3Rc/3Rc （PANC-1）.The optical density ratio of homozygous 3Rg/3Rg cells and homozygous 3Rc/3Rc cells was 1.723±0.062 and 1.063±0.134,respectively,and this difference was statistically significant （P 〈0.05）.Cells with the 2R/2R and 2R/3R genotypes of TS were hypersensitive to 5-FU in vitro as compared with those with the 3R/3R cells.Conclusions Polymorphisms in the TS gene influenced its protein expression and affected sensitivity of 5-FU in seven pancreatic cancer cell lines.Cells with the 3R/3R genotype had higher TS protein expression than the 2R/2R or 2R/3R genotypes.Cells of the 3R/3R genotype with high TS protein expression were shown lower 5-FU sensitivity than cells with the 2R/2R or 2R/3R genotypes.These data warrant large-scale clinical studies to assess the role of polymorphisms in the TS gene on its protein expression and chemosensitivity to 5-FU in pancreatic cancer.

Thymidylate synthase genetic polymorphisms and cancer risk： a meta-analysis of 37 case-control studies

Background Several studies have evaluated the association between polymorphisms of thymidylate synthase （TS） and cancer risk in diverse populations but with conflicting results. By pooling the relatively small samples in each study, it is possible to evaluate the association using a meta-analysis. Methods A comprehensive search was conducted to identify all case-control studies on TS on a 28-bp tandem repeats in 5＇untranslated region （5UTR） and a 6-bp insertion （ins） and deletion （del） mutation in 3＇UTR of the gene and cancer risk. Meta-analysis was conducted using a fixed and random effect model. Results Our meta-analysis on a total of 13307 cancer cases and 18226 control subjects from 37 published case-control studies showed no significant association between the risk of cancer and the 5＇UTR 28-bp tandem repeats polymorphism （3R/3R vs. 2R/2R： 0R=1.06, 95% CI, 0.93-1.20） or the 3＇UTR 6-bp ins/del polymorphism （del6/del6 vs. ins6/ins6： OR=0.93, 95% CI, 0.81-1.08） with significant between-study heterogeneity. In the cancer type- and ethnic subgroup-stratification analyses, we did not find any association between TS polymorphisms and cancer risk either. Conclusion TS 5＇UTR 28-bp tandem repeats and 3＇UTR 6-bp ins/del polymorphisms may not be associated with cancer risk.

Integrating TYMS, KRAS and BRAF testing in patients with metastatic colorectal cancer

AIM To investigate the impact of thymidylate synthase(TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer(m CRC) patients treated with chemotherapy. METHODS Clinical data were collected retrospectively from records of consecutive patients with m CRC treated with fluoropyrimidine-based chemotherapy from 1/2005 to 1/2007. Formalin-fixed paraffin-embedded tissues were retrieved for analysis. TYMS genotypes were identified with restriction fragment analysis PCR, while KRAS and BRAF mutation status was evaluated using real-time PCR assays. TYMS gene polymorphisms of each of the 3' untranslated region(UTR) and 5'UTR were classified into three groups according to the probability they have for high, medium and low TYMS expression(and similar levels of risk) based on evidence from previous studies. Univariate and multivariate survival analyses were performed.RESULTS The analysis recovered 89 patients with m CRC(46.1% de novo metastatic disease and 53.9% relapsed). Of these, 46 patients(51.7%) had colon cancer and 43(48.3%) rectal cancer as primary. All patients were treated with fluoropyrimidine-based chemotherapy(5FU or capecitabine) as single-agent or in combination with irinotecan or/and oxaliplatin or/and bevacizumab. With a median follow-up time of 14.8 mo(range 0-119.8), 85 patients(95.5%) experienced disease progression, and 63 deaths(70.8%) were recorded. The 3-year and 5-year OS rate was 25.4% and 7.7% while the 3-year progression-free survival rate was 7.1%. Multivariate analysis of TYMS polymorphisms, KRAS and BRAF with clinicopathological parameters indicated that TYMS 3'UTR polymorphisms are associated with risk for disease progression and death(P < 0.05 and P < 0.03 respectively). When compared to tumors without any del allele(genotypes ins/ins and ins/loss of heterozygosity(LOH) linked with high TYMS expression) tumors with del/del genotype(low expression group) and tumors with ins/del or del/LOH(intermediate expression group) have lower risk for disease progression(HR = 0.432, 95%CI: 0.198-0.946, P < 0.04 and HR = 0.513, 95%CI: 0.287-0.919, P < 0.03 respectively) and death(HR = 0.366, 95%CI: 0.162-0.827, P < 0.02 and HR = 0.559, 95%CI: 0.309-1.113, P < 0.06 respectively). Additionally,KRAS mutation was associated independently with the risk of disease progression(HR = 1.600, 95%CI: 1.011-2.531, P < 0.05). The addition of irinotecan in 1st line chemotherapy was associated independently with lower risk for disease progression and death(HR = 0.600, 95%CI: 0.372-0.969, P < 0.04 and HR = 0.352, 95%CI: 0.164-0.757, P < 0.01 respectively).CONCLUSION The TYMS genotypes ins/ins and ins/LOH associate with worst prognosis in m CRC patients under fluoropyrimidine-based chemotherapy. Large prospective studies are needed for validation of our findings.

Molecular determinants of response to 5-fluorouracil-based chemotherapy in colorectal cancer: The undisputable role of microribonucleic acids

5-flurouracil(5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer(CRC).Despite significant progress in the treatment of CRC during the last decades,5-FU drug resistance remains the most important cause of failure in CRC therapy.Resistance to 5-FU is a complex and multistep process.Different mechanisms including microsatellite instability,increased expression level of key enzyme thymidylate synthase and its polymorphism,increased level of 5-FU-activating enzymes and mutation of TP53 are proposed as the main determinants of resistance to 5-FU in CRC cells.Recently,microribonucleic acids(miRNA)and their alterations were found to have a crucial role in 5-FU resistance.In this regard,the miRNA-mediated mechanisms of 5-FU drug resistance reside among the new fields of pharmacogenetics of CRC drug response that has not been completely discovered.Identification of the biological markers that are related to response to 5-FU-based chemotherapy is an emerging field of precision medicine.This approach will have an important role in defining those patients who are most likely to benefit from 5-FU-based chemotherapy in the future.Thereby,the identification of 5-FU drug resistance mechanisms is an essential step to predict and eventually overcome resistance.In the present comprehensive review,we will summarize the latest knowledge regarding the molecular determinants of response to 5-FU-based chemotherapy in CRC by emphasizing the role of miRNAs.

Review of 5-FU resistance mechanisms in colorectal cancer:clinical significance of attenuated on-target effects

The emergence of chemoresistant disease during chemotherapy with 5-Fluorouracil-based(5-FU-based)regimens is an important factor in the mortality of metastatic CRC(mCRC).The causes of 5-FU resistance are multifactorial,and besides DNA mismatch repair deficiency(MMR-D),there are no widely accepted criteria for determining which CRC patients are not likely to be responsive to 5-FU-based therapy.Thus,there is a need to systematically understand the mechanistic basis for 5-FU treatment failure and an urgent need to develop new approaches for circumventing the major causes of 5-FU resistance.In this manuscript,we review mechanisms of 5-FU resistance with an emphasis on:(1)altered anabolic metabolism limiting the formation of the primary active metabolite Fluorodeoxyuridylate(5-Fluoro-2'-deoxyuridine-5'-O-monophosphate;FdUMP);(2)elevated expression or activity of the primary enzymatic target thymidylate synthase(TS);and(3)dysregulated programmed cell death as important causes of 5-FU resistance.Importantly,these causes of 5-FU resistance can potentially be overcome through the use of next-generation fluoropyrimidine(FP)polymers(e.g.,CF10)that display reduced dependence on anabolic metabolism and more potent TS inhibitory activity.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate syn- thase activity which is responsible for the C-5 methyla- tion of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 A, 1.76 A and 2.28 A resolutions, respec- tively. Loop 1 （residues 117-131）, Loop 2 （residues 192- 201） and the substrate recognition peptide （residues 94- 102） of PolB exhibit considerable conformational flexi-bility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methy-lase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.