FOXM1 inhibitor, Siomycin A, synergizes and restores 5-FU cytotoxicity in human cholangiocarcinoma cell lines via targeting thymidylate synthase.

AIMS: 5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. MAIN METHODS: Human CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. KEY FINDING: Single drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. SIGNIFICANCE: Targeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.

Standing the test of time: targeting thymidylate biosynthesis in cancer therapy.

Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.

Ubiquitin-independent proteasomal degradation.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity.

BACKGROUND: Fluoropyrimidine drugs are extensively used for the treatment of solid cancers. However, adverse drug reactions are a major clinical problem, often necessitating treatment discontinuation. The aim of this study was to identify pharmacogenetic markers predicting fluoropyrimidine toxicity. METHODS: Toxicity in the first four cycles of 5-fluorouracil or capecitabine-based chemotherapy were recorded for a series of 430 patients. The association between demographic variables, DPYD, DPYS, TYMS, MTHFR, CDA genotypes, and toxicity were analysed using logistic regression models. RESULTS: Four DPYD sequence variants (c.1905+1G>A, c.2846A>T, c.1601G>A and c.1679T>G) were found in 6% of the cohort and were significantly associated with grade 3-4 toxicity (P<0.0001). The TYMS 3'-untranslated region del/del genotype substantially increased the risk of severe toxicity (P=0.0123, odds ratio (OR)=3.08, 95% confidence interval (CI): 1.38-6.87). For patients treated with capecitabine, a MTHFR c.1298CC homozygous variant genotype predicted hand-foot syndrome (P=4.1 × 10⁻6, OR=9.99, 95% CI: 3.84-27.8). The linked CDA c.-92A>G and CDA c.-451C>T variants predicted grade 2-4 diarrhoea (P=0.0055, OR=2.3, 95% CI: 1.3-4.2 and P=0.0082, OR=2.3, 95% CI: 1.3-4.2, respectively). CONCLUSION: We have identified a panel of clinically useful pharmacogenetic markers predicting toxicity to fluoropyrimidine therapy. Dose reduction should be considered in patients carrying these sequence variants.

Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence.

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

MicroRNA expression profile associated with response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients.

BACKGROUND: Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients. METHODS: We selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling. RESULTS: Expression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified. CONCLUSIONS: Our pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

BACKGROUND/AIMS: The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. METHODOLOGY: Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. RESULTS: TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. CONCLUSIONS: TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

[A brief overview of a lung cancer biomarker: thymidylate synthase]. / Un regard simple sur la biologie moléculaire du cancer bronchique : la thymidylate synthase (TS).

Thymidylate synthase (TS) is an enzyme, which catalyzes the methylation of deoxyuridylate to deoxythymidylate using 5.10-methylenetetrahydrofolate as a cofactor. For this reason, TS has been widely investigated and is one of the best-known drug targets in the anticancer area. Antimetabolites have been developed to target TS and among them, pemetrexed is now considered as part of the standard treatment for lung cancer and mesothelioma. Intratumoral expression of TS mRNA has been shown to be associated with prognosis and with the response to 5-FU therapy in patients with breast, colorectal, head and neck cancer types. Recent findings suggest that TS might be a biomarker for NSCLC treated with pemetrexed, as lower response rates in squamous cell carcinoma and small cell carcinoma may be due to a higher expression of TS. Specific validation for this use as a biomarker is awaited. All these recent findings suggest that TS could be a useful predictive marker of the treatment efficacy of antifolate drugs and indicate that both Real-Time PCR and immuno-histochemistry might be used to assess TS expression levels. This may help in defining the best therapeutic strategy.

Defining the role of mutations in Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene using an episomal Plasmodium falciparum transfection system.

Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells.

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

The role of molecular markers in predicting response to therapy in patients with colorectal cancer.

Advances in systemic therapy for colorectal cancer have dramatically improved prognosis. While disease stage has traditionally been the main determinant of disease course, several molecular characteristics of tumor specimens have recently been shown to have prognostic significance. Although to date no molecular characteristics have emerged as consistent predictors of response to therapy, retrospective studies have investigated the role of a variety of biomarkers, including microsatellite instability, loss of heterozygosity of 18q, type II transforming growth factor beta receptor, thymidylate synthase, epidermal growth factor receptor, and Kirsten-ras (KRAS). This paper reviews the current literature, ongoing prospective studies evaluating the role of these markers, and novel techniques such as gene profiling, which may help to uncover the more complex molecular interactions that will predict response to chemotherapy in patients with colorectal cancer.

YjjG, a dUMP phosphatase, is critical for thymine utilization by Escherichia coli K-12.

Exogenous thymine must be converted to thymidine to enable a thyA (thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A yjjG thyA mutant cannot use thymine for growth on a glucose minimal medium.

Genomic copy number changes affecting the thymidylate synthase (TYMS) gene in cancer: a model for patient classification to aid fluoropyrimidine therapy.

Thymidylate synthase (TS) is an important target for 5-fluorouracil (5FU)-based therapy. The TS polymorphic 5'-untranslated region tandem repeat sequence is under investigation to guide 5FU treatment, yet current protocols omit consideration of copy number changes at the TS locus. We surveyed the TS tandem repeat sequence and found copy number changes in gastrointestinal cancers. Ten of 12 informative cases had loss of heterozygosity (LOH), whereas two others and an additional cell line had a novel TS genotype, allelic imbalance at the TS locus due to polysomy. Experimentally, we studied a diploid colorectal cancer line heterozygous at TS to mimic three common TS genotypes of cancers. Using genetic engineering, we deleted the short tandem repeat (two repeats) allele and retained the long (three repeats) allele to produce artificial LOH at the TS gene; the TS(+/-) line had a reduced TS protein expression and was hypersensitive to 5FU and 5-fluoro-2'-deoxyuridine in vitro as compared with syngeneic control lines. We linked this sensitivity directly to the reduced TS expression by introducing exogenous TS cDNA expression into the TS(+/-) line (i.e., increased TS copies). Our model predicts that the 5FU sensitivity of a tumor is modified by aneuploidy producing copy number changes of TS alleles by one or more of the following: LOH, amplification, and, as presented here, copy number changes due to polysomy. The data suggest that TS copy number in a patient's tumor may be a dominating variable affecting 5FU responsiveness.

Prediction of clinical outcome of fluoropyrimidine-based chemotherapy for gastric cancer patients, in terms of the 5-fluorouracil metabolic pathway.

Fluoropyrimidines are widely used in chemotherapy regimens for metastatic gastric cancer. Interindividual variation in the enzyme activity of the 5-fluorouracil (FU) metabolic pathway can affect the extent of 5-FU metabolism and affect the efficacy of 5-FU based chemotherapy. In this review, the role of the genetic factors affecting the therapeutic efficacy of fluoropyrimidines is discussed, with a special emphasis on enzymes involved in the 5-FU metabolic pathway. The gene expressions of thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase are discussed in relation to the efficacy of fluoropyrimidine treatment for metastatic gastric cancer. These candidate genes, along with others yet to be identified, could allow accurate prediction of the clinical outcome in patients receiving fluoropyrimidine-based chemotherapy in the future. Well-designed and large prospective studies, which include relevant pharmacogenetic parameters, are needed to confirm the values required to predict clinical outcome.

Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Cyclin D1 antisense oligodexoyneucleotides inhibits growth and enhances chemosensitivity in gastric carcinoma cells.

AIM: To examine the effects of cyclin D1 antisense oligodexoyneucleotides (ASODN) on growth and chemosensitivity of gastric carcinoma cell lines SGC7901 and its mechanism. METHODS: Phosphorothioate modified cyclin D1 ASODN was encapsulated by LipofectAMINE2000 (LF2000) and transfected into cells, the dose-effect curves and growth curves were observed. 5-FU, MTX, CDDP of different concentrations were given after transfecting cells with cyclin D1 ASODN for 24 h the dose-effect responses were observed and IC50s were calculated. The mRNA expression of cyclin D1, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR) was detected by reverse transcription-PCR (RT-PCR) at 24 h and 48 h after transfection. RESULTS: Dose-dependent inhibitory effect was caused by cyclin D1 ASODN in SGC7901 cells. Transfecting gastric carcinoma cells with 0.2 micromol/L cyclin D1 ASODN for 24 h could inhibit growth significantly and reduce expression of cyclin D1 mRNA. Cyclin D1 ASODN could increase the chemosensitivity to 5-FU, MTX, CDDP in cells. The IC50s of different chemotherapeutic agents in ASODN plus chemotherapy groups were significantly lower than those in controls. Transfection with cyclin D1 ASODN leaded to an increase in TS and DHFR mRNA and a decrease in TP mRNA as determined by RT-PCR at 24 h, the alterations were more significant at 48 h. CONCLUSIONS: Cyclin D1 ASODN can decrease mRNA expression of cyclin D1, inhibit growth and enhance the chemosensitivity by changing the expression of enzymes related to metabolism of chemotherapeutic agents in SGC7901 gastric carcinoma cells.

Introduction of an initiator element in the mouse thymidylate synthase promoter alters S phase regulation but has no effect on promoter bidirectionality.

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element, is bidirectional and initiates transcription at multiple start sites across broad initiation windows upstream and downstream of the 30 nt essential promoter region. The TS promoter also plays an essential role in the post-transcription regulation of TS gene expression during the G(1)-S phase transition. The goal of this study was to determine if the addition of a TATAA box or an initiator element would have a significant effect on start-site pattern, promoter bidirectionality and S phase regulation of the TS gene. A TATAA box and/or an initiator element were inserted downstream of the TS essential promoter region, and the modified promoters were used to drive expression of indicator genes. The engineered genes were transfected into cultured mammalian cells, and the effects of the mutations were determined. Addition of the TATAA box and especially the initiator element had a significant effect on the transcription start site pattern, indicating that the elements were functional. Unexpectedly, addition of one or both of these elements had no effect on promoter bidirectionality. However, inclusion of the initiator element led to a significant reduction in S phase regulation of TS mRNA levels, indicating that changes in promoter architecture can perturb normal S phase regulation of TS gene expression.