Thymidylate Synthase Protein Expression Research Articles

The Predictive Value of ERCC1, RRM1, and Thymidylate Synthase in Advanced Malignant Pleural Mesothelioma Patients Treated with Platinum-Based Chemotherapy.

A rare yet severe neoplasia called malignant pleural mesothelioma (MPM) typically manifests itself in advanced stages. Despite some improvements in the treatment of patients with MPM, this malignancy continues to have a detrimental prognosis . The primary goal of the present study was to assess the associatin between ERCC1, RRM1, and thymidylate synthase (TS) expression and disease outcome in patients with malignant pleural mesothelioma (MPM) treated with either pemetrexed plus cisplatin or gemcitabine plus cisplatin. This prospective cohort study was done on ninety-one advanced MPM patients. The patients received either pemetrexed plus platinum (55 of 91) or gemcitabine plus platinum chemotherapy (36 of 91). Tissue biopsy was taken at time of diagnosis. Immunohistochemistry was used to assess the levels of ERCC1, RRM1, and TS transcription in tissue biopsies (paraffin embedded). Based on the findings, 70% of patients with low expression of TS had low expression of ERCC1, and 68% of patients with high expression of TS had high expression of ERCC1, suggesting a significat association between ERCC1 expression and TS expression (p=0.005). However, there was no relation between ERCC1 and RRM1 expression patterns (p= 0.296). In patients underwen platinum-based theraphy (n 91), a significant correlation was detected between low ERCC1 median High-scoring and longer progression time (TTP;9.6 v 5.3 months;P< .001) or overall survival (OS) (OS;18.1 v 9.1 months; P<0.001). A significant correlation was found between low TS protein expression and longer time progression (TTP; 11.8 v 5.4 months; P< .001) or OS (OS; 19.8 v 8.5 months; P <0.001) in patients undergoing pemetrexed plus platinum chemotherapy (n 55). Low RRM1 expression was accompanied by high progression free survival (TTP; 10.6 v 3.8 months) and OS (OS; 20.6 v 8.6 months) in patients receiving gemcitabine plus platinum chemotherapy. Based on the multivariate test results, the independent variables significantly affecting OS were tumor stage (HR: 2.3; 95% CI: 1.1-4.9; p= 0.021) and ERCC1 (HR: 2.7; 95% CI: 1.7-4.3; p < 0.001). Decreased TS protein expression can be indicative of greater responsivness to pemtrexed and of longer TTP and OS in individuals with advanced MPM (locally progressed or metastatic) who are receiving pemetrexed-based chemotheraphy. Low ERCC1 expressions in individuals with advanced MPM can predict increased PFS and OS, as well as a better responsivness to platinum-based chemotherapy. In patients with progressed MPM receiving gemcitabine plus cisplatin chemotherapy, lower RRM1 expression was associated with a better prognosis, longer PFS, and longer OS.

Targeting Thymidylate Synthase and tRNA-Derived Non-Coding RNAs Improves Therapeutic Sensitivity in Colorectal Cancer.

Some colorectal cancer (CRC) patients are resistant to 5-fluorouracil (5-FU), and high expression levels of thymidylate synthase (TS) contribute to this resistance. This study investigated whether quercetin, a representative polyphenol compound, could enhance the effect of 5-FU in CRC cells. Quercetin suppressed TS levels that were increased by 5-FU in CRC cells and promoted the expression of p53. Quercetin also induced intracellular and mitochondrial reactive oxygen species (ROS) production and Ca2+ dysregulation in a 5-FU-independent pathway in CRC cells. Furthermore, quercetin decreased mitochondrial membrane potential in CRC cells and inhibited mitochondrial respiration. Moreover, quercetin regulated the expression of specific tiRNAs, including tiRNAHisGTG, and transfection of a tiRNAHisGTG mimic further enhanced the apoptotic effect of quercetin in CRC cells. An enhanced sensitivity to 5-FU was also confirmed in colitis-associated CRC mice treated with quercetin. The treatment of quercetin decreased survival rates of the CRC mouse model, with reductions in the number of tumors and in the disease activity index. Also, quercetin suppressed TS and PCNA protein expression in the distal colon tissue of CRC mice. These results suggest that quercetin has the potential to be used as an adjuvant with 5-FU for the treatment of CRC.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Micro-RNA-215 and -375 regulate thymidylate synthase protein expression in pleural mesothelioma and mediate epithelial to mesenchymal transition

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM.

Association of expression and genotypes of thymidylate synthase in non-small cell lung cancer patients with different clinicopathological characteristics

Abstract Objective To explore the expression and genotypes of thymidylate synthase (TS) in patients of non-small cell lung cancer (NSCLC) with different clinicopathological characteristics. Methods The expression profiles of TS were examined by immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 160 patients with NSCLC. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect TS-5′UTR tandem repeats, G/C nucleotide polymorphisms, and 3′UTR 6 bp deletion/insertion polymorphisms. The relationships between clinicopathological characteristics and TS expression or genotypes were investigated through χ 2 test. Kaplan–Meier survival analysis was used to analyze the association between TS expression and overall survival (OS) and disease-free survival (DFS) of NSCLC patients. Results The expression levels of TS protein and TS gene in NSCLC tissues were significantly higher than that in paracancerous tissues (P &lt; 0.05). Furthermore, high expression of TS protein and 5′UTR polymorphism of TS gene showed significant correlation with differentiation, TNM stage, and lymph node metastases. The frequency of −6 bp/−6 bp genotypes in patients with NSCLC was 43.13% (69/160), which was higher than others. In addition, the rate of TS protein overexpression in NSCLC patients with 3R/3R was 79.79%, which was higher than others. Interestingly, high expression of TS protein predicted shorter DFS and OS and lower 3-year DFS rate and 3-year OS rate. Conclusions The expression levels of TS in NSCLC were significantly increased and may help to predict the prognosis of NSCLC, and high expression of TS protein and 5′UTR polymorphism of TS gene were significantly related to differentiation, TNM stage, and lymph node metastases.

Prognostic and Predictive Role of Excision Repair Cross-complementation Group 1 and Thymidylate Synthase in Colorectal Carcinoma Patients Received FOLFOX Chemotherapy: An Immunohistochemical Study.

We aim to determine the frequency of thymidylate synthase (TS) and excision repair cross-complementation group 1 (ERCC-1) immunohistochemical (IHC) expression and its relationship with clinicopathologic variables in colorectal carcinoma (CRC) patients. In addition, we aim to assess the correlation between TS and ERCC-1 expression and the response of these cases to oxaliplatin and 5-fluorouracil chemotherapy (FOLFOX). Fifty-one CRC patients were prepared for IHC analysis of ERCC-1 and TS protein expression. All patients received oxaliplatin and 5-fluorouracil combined chemotherapy (FOLFOX) and were followed up for 24 months. The data analysis showed that high ERCC-1 and TS expression was significantly associated with early treatment failure (P=0.020 and 0.000). In contrast, TS immunoexpression affects the disease-free survival rate (P=0.010). The presence of deep tumor invasion, distant metastasis, lymph node metastasis, and high Dukes' classification were significantly statistically associated with early treatment failure (P=0.001, 0.000, 0.041, and 0.015, respectively). Our results showed that both ERCC-1 and TS are predictive factors for early treatment failure in CRC patients. TS protein is a prognostic factor for disease-free survival rates. This supports the theory that both ERCC-1 and TS can be used to improve chemotherapeutic outcomes in CRC patients. High expression of TS and ERCC-1 helps in the identification of cases that will get fewer benefits from FOLFOX chemotherapy. As an innovative strategy, in these cases, we can use alternative chemotherapeutic regimens or add an extra agent. In addition, Dukes' classification and lymph node metastasis are predictive factors for early treatment failure. Thus, all those values can be used to predict CRC patients with bad prognosis and those who will get fewer benefits from FOLFOX.

Abstract C059: Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation

Abstract Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. NUC-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on NUC-3373 and the role of NUC-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating NUC-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and NUC-3373. Thymidine supplementation was used to indirectly assess the effect of NUC-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments NUC-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following NUC-3373 treatment. Supplementation with exogenous thymidine rescued cells from NUC-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for NUC-3373. In selected cell lines treated with NUC-3373, TS ternary complexes were detected for at least 72 hours. Treatment with NUC-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received NUC-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, NUC-3373 was not catabolised by DPD, highlighting the ability of NUC-3373 to bypass a key rate-limiting factor associated with 5-FU. NUC-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that NUC-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of NUC-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059

Abstract 2082: NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines

Abstract BACKGROUND: Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). Although widely studied, there are mixed reports as to whether high or low basal tumor TS levels are prognostic of response to 5-FU therapy. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. This study aimed to determine the prognostic value of cancer cell TS protein expression in predicting therapeutic response to NUC-3373 and to further explore the effects of NUC-3373 on TS biology. METHODS: IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. In these two cell lines, endogenous, induced, and ternary complex TS expression was measured by Western blot analysis and the cellular localization of TS was confirmed by immunocytochemistry and assessed by stereological methods. RESULTS: TS protein expression was measured in nine CRC cell lines treated with NUC-3373 and no correlation was found between TS protein levels and the IC50 for NUC-3373. This indicates that the sensitivity of these cell lines to NUC-3373 is not dependent on basal TS protein expression levels. When the two cell lines selected for their sensitivity/resistance to NUC-3373 were treated with NUC-3373, TS expression was induced after 12 hours and remained elevated at 48 hours. In addition, treatment with NUC-3373 was associated with a translocation of TS from the nucleus to the cell cytoplasm, which was more pronounced in the NUC-3373 sensitive cells. Translocated TS did not localize to vesicles or the Golgi apparatus; the cytoplasmic localization remains to be determined. CONCLUSIONS: These results suggest that basal TS levels are not prognostic of response to NUC-3373 in CRC cells, instead NUC-3373 induces TS expression and causes nuclear to cytoplasmic translocation. This novel finding is indicative of an alternative mechanism of action for FUDR-MP in promoting anti-cancer activity that is independent of the DNA damage pathway. Citation Format: Fiona G. McKissock, In Hwa Um, Peter Mullen, David J. Harrison. NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2082.

PM2.5 induces cell cycle arrest through regulating mTOR/P70S6K1 signaling pathway.

Fine particulate matter (PM2.5) pollution has become a serious problem in China. This study aims to elucidate the toxicity mechanism of PM2.5. Protein levels were detected by western blotting and RT-qPCR, and cell cycle was detected by flow cytometry. The results showed that exposure to PM2.5 induces cell cycle arrest and downregulation of the expression of cyclin D1 protein. Moreover, the protein expression of thymidylate synthase (TS) enzyme was found to be downregulated and the mRNA expression of TS was upregulated after PM2.5 exposure. Knockout of TS gene promoted cell cycle arrest and downregulation of the expression of cyclin D1 protein after PM2.5 exposure. Our data further revealed that PM2.5 exposure downregulates the expression of TS and cyclin D1 partially through the downregulation of the mammalian target of rapamycin (mTOR)/P70S6K1 signaling pathway. Thus, these findings indicate that PM2.5-induced cell cycle arrest might be due to the downregulation of mTOR/P70S6K1 signaling pathway, and thus inhibits the expression of TS protein.

RESEASRCH POGRESS FOR THYMIDYLATE SYNTHASE AND INHIBITORS IN CANCERS COUPLE IN CHEMICAL STRUCTURES

Cancer new drug development and testing mostly rely on existing drug which plays a great role as templates, and the biomarkers. Thymidylate Synthase Inhibitors (TSI) tabulated below are available for the diseases conditions like cancer which encircle the thymidylate synthase (TS). Sparse data are available for TS involvement in cancer. TS is prudent in the synthesis of thymidylate which is useful for body functionalities, and its production pathways are observed to contain supportive proteins. The loss of thymine in cancerous cells couple in abnormal DNA synthesis influenced cell apoptosis. An increased TS protein expression seen in many cancers, serving as aggressive phenotype. Cancers is TS disease, still without a cure, taking away the lives of at least 8 million people every year irrespective of nationality, remained a burden that let world health organization to request for urgent treatment. The trouble encompassing cancer in general and that which generated from TS is the principal base of this review. Although, it has been used in other disease and have not yield total cure in cancer yet, TSI potentially utilized to offset the problem responsible for low circulated thymidine in human plasma, is useful to manage cancer disease. Members of TSI bears five or six cycle ring in their structure. The pterine and pyrimidine are compounds which are incorporation with another compound by utilizing the technique of nucleophilic displacement, multicomponent condensation and ultimate combination principle, molecular hybridization strategy and the structural activity relationship mechanism to develop new small TS inhibitors. Through the available English articles about TS related cancer and TSI in ScienceDirect database are utilized here. The search was made by key in the keywards singularly and in pairs to present this comprehensive knowledge about TSI inhibitors, and evaluate TS enzyme mechanisms which contributes to cancer and how the TSI can circumvent cancer disease. Conclusively, the series of TSI utilized the TS protein enzyme to ombat TS human cancer, are also in use as a fundamental molecular template for new drug. TS activity can produce the novel thymidylate. This work will be of prudent to researchers in drug design and synthesis and as well to hospital clinician to support the judgement inside cancer disease treatment.

The relationship between the expression of thymidylate synthase, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase, excision repair cross-complementation group 1 and class III β-tubulin, and the therapeutic effect of S-1 or carboplatin plus paclitaxel in non-small-cell lung cancer.

Previous studies have reported that the expressions of specific proteins may predict the efficacy of chemotherapy agents for non-small cell lung cancer (NSCLC) patients. The present study evaluated the expression of proteins hypothesized to be associated with the effect of chemotherapeutic agents in 38 NSCLC patients with pathological stage II and IIIA. The subjects received carboplatin plus paclitaxel (CP) or S-1 as adjuvant chemotherapy following complete resection. The protein expressions evaluated were those of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phsphoribosyltransferase (OPRT), which were suspected to be associated with the effect of S-1 agents, excision repair cross-complementation group 1 (ERCC1), which was suspected to be associated with the effect of platinum-based agents, and class III β-tubulin (TUBB3), which was suspected to be associated with the effect of taxane-based agents. The positive rate of TS was 55.3% (n=21/38), DPD was 57.9% (n=22/38), OPRT was 42.1% (n=16/38), ERCC1 was 47.4% (n=18/38) and TUBB3 was 44.7% (n=17/38). Among the patients who received S-1 adjuvant chemotherapy, TS-negative cases demonstrated a significantly better disease-free survival than positive cases. Thus, TS protein expression may have been a factor that predicted the effect of S-1 agent as adjuvant chemotherapy.

A study of thymidylate synthase gene expression as a biomarker for the treatment of esophageal adenocarcinoma.

e15563 Background: Expression of thymidylate synthase (TS), a target of fluoropyrimidine-based (5-FU) chemotherapy, has prognostic value in colorectal adenocarcinoma. Its value as a biomarker in treatment of esophageal adenocarcinoma (EAC) is unknown. With widespread adoption of CROSS trial protocol for treatment of EAC, 5-FU is frequently omitted from regimens. We retrospectively analyzed the association between TS protein expression and clinical outcomes in EAC. Methods: TS expression levels were measured by immunohistochemistry in formalin-fixed paraffin-embedded specimens from 35 primary EAC patients from 2 institutions (range 5/2006-1/2016); at time of diagnosis and surgical resection (33 neoadjuvant chemoradiation and 2 surgery alone). TS expression higher than 1+, 5% was read as positive. Mean follow up was 1323 days (range 263-3677). TS expression correlated with treatment response and survival in patients treated with and without 5-FU therapy (log-rank test, Kaplan-Meier method). Results: In the cohort of 35 patients, 21 patients (60%) had pretreatment positive TS expression. Overall survival (OS) for TS negative patients was 1216 days versus 974 days for TS positive (p = 0.15). In the 23 patients treated with 5-FU, TS-negative patients had a trend towards longer overall survival compared to TS-positive patients, mean OS (1272 days versus 743 days, p = 0.08). In the group of 12 patients not treated with 5-FU, 2 patients showed negative TS staining. Neoadjuvant chemoradiation was performed in 33/35 patients. Downstaging was observed in 69% (9/13) of patients with negative pre-treatment TS levels versus 55% (11/20) in pre-treatment TS positive patients (p = 0.48). In 23 patients treated with 5FU, 9/12 (75%) were downstaged in the TS-negative cohort versus 6/11 (45%) in the TS-positive cohort (p = 0.21). In the 5FU treatment group, complete response was seen in 2 patients with negative TS staining. Conclusions: Low TS expression in EAC patients trended towards improved survival and tumor downstaging, particularly in patients treated with neoadjuvant 5-FU and platinum based therapy. Prospective data and greater patient numbers may be necessary to demonstrate statistical significance.

Protein and mRNA expression of folic acid-associated enzymes as biomarkers for the cytotoxicity of the thymidylate synthase-targeted drugs, pemetrexed and S-1, in non-small cell lung cancer.

The thymidylate synthase (TS)-targeted drugs, pemetrexed and S-1, exert an important role in advanced non-small cell lung cancer (NSCLC) treatment; folic acid-associated enzymes are expected to behave as biomarkers, although their role has yet to be fully elucidated. In the present study, a single-institutional prospective analysis, in which the mRNA and protein expression levels of five folic acid-associated enzymes were evaluated with surgical specimens of NSCLC, was performed. Drug sensitivity was evaluated using a collagen gel droplet-embedded culture drug sensitivity test (CD-DST) in vitro. A total of 50 patients with NSCLC were enrolled, and the mRNA and protein expression levels of five enzymes were assessed in 47 and 46 patients, respectively. A significant association was identified between mRNA and protein expression in TS (r=0.6266), but the correlation between mRNA and protein expression levels for the other four enzymes was poor. TS mRNA expression was significantly higher in poorly differentiated tumors compared with moderately differentiated tumors (P=0.0399). TS protein expression was significantly higher in patients with pleural invasion or lymphatic invasion compared with those lacking them (P=0.027 and 0.030, respectively). CD-DST revealed that none of the tumors that were sensitive to pemetrexed, but not to S-1, were well differentiated, whereas none of the tumors that were sensitive to S-1, but not to pemetrexed, were poorly differentiated. More prominent vascular invasion was observed in the tumors that were sensitive to S-1. The only factors that exhibited the potential to discriminate the cytotoxicity of pemetrexed from S-1 were tumor differentiation grade and vascular invasion.

Influence of thymidylate synthase expression on survival in patients with colorectal cancer

Background: Thymidylate synthase (TS) plays a critical role in nucleotide metabolism and is an important target for 5-fluorouracil (5-FU), the standard chemotherapeutic drug for treatment of colorectal cancer (CRC). Aims and Methods: The present study aimed to evaluate TS variable number tandem repeat sequences (VNTR) polymorphism by polymerase chain reaction and TS protein expression by immunohistochemistry and its association with clinicopathological parameters in untreated CRC patients (n = 100). Further, the prognostic and predictive role of TS has been evaluated. Results: For TS VNTR polymorphism, the observed frequencies of 2R/2R, 2R/3R, and 3R/3R genotypes were 22%, 51%, and 27%, respectively. When immunohistochemical localization was studied, cytoplasmic staining for TS was observed in 70% of patients. A significant inverse correlation was noted between TS protein expression and tumor, node, metastasis staging (P = 0.027), Dukes' staging (P = 0.039), and lymph node status (P = 0.012) of CRC patients. However, there was no significant correlation between TS VNTR polymorphism and TS protein expression. On survival analysis, a significantly shorter overall survival (OS) was seen in CRC patients with negative protein expression (P = 0.031). Moreover, the subgroup of CRC patients treated only with surgery also showed a trend of poor OS in patients with negative TS protein expression (P = 0.058). However, neither TS polymorphism nor its protein expression was able to predict relapse-free survival. Conclusion: Negative TS protein expression may be related to unfavorable clinical outcome in CRC patients. However, further studies in a larger set of patients are necessary to better assess TS as a prognostic and predictive marker for 5-FU response in CRC patients.

Abstract 2159: In vitro characterization of PTAU and FU interactions in colon cancer cells

Abstract Background: Fluorouracil (FU) remains a main anticancer therapy for colorectal cancer (CRC) and other solid tumors. The mechanism of action of FU is associated with inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. High toxicity and wide-spread chemoresistance, however, have limited the utilization of FU-based therapies. Five intracellular enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP), and uridine phosphorylase (UP) are key determinants of FU sensitivity or resistance. Combination therapies aimed at improving the bioavailability of FU and reducing host toxicity has been evaluated. Recently, our group developed a new uridine phosphorylase inhibitor, 5-phenylthio-acyclouridine (PTAU) that has shown promise in reducing host toxicity and increasing FU efficacy in vivo. The current studies were to characterize interactions of PTAU and FU in vitro and to assess its effects on key metabolic enzymes in human colon cancer cells. Hypothesis: We hypothesized that inhibition of uridine phosphorylase by PTAU would ameliorate the toxic effects of FU and enhance its efficacy by permitting increased doses of FU. Methods: Three colon cancer cell lines with differing p53 status, HCT116-p53wt (wild-type), HCT116-p53null, and HT-29 (p53-mutant); and a normal colonic epithelial cell line, CRL-1790, were used. Doubling times were determined, and IC50 values for FU were derived from kill curves using MTT assay. Cells were treated for 72 hours with IC50 doses of FU, PTAU (100 μM) or uridine (25 μM), alone or in combination, or with the solvent, DMSO. mRNA levels for DPD, TS, TP, and UP were quantitated by qRT-PCR; for intracellular protein expression, cells were fixed and stained with monoclonal antibodies and analyzed by flow cytometry. Results: FU sensitivity correlated with cell doubling times. Basal expression of FU metabolic enzymes differed between CRC and normal cells. mRNAs for DPD and UP were higher in normal relative to cancer cells, but TS mRNA was higher in cancer cells. FU treatment upregulated mRNA expression of DPD, UP, and TS in cancer cells but not in normal cells. Only in cancer cells, DPD induction by FU was potentiated by PTAU. In contrast, treatment with FU decreased protein levels of DPD, TP, and TS in cancer cells. PTAU induced TS protein expression in cancer cells, an effect that was reduced by co-treatment with FU. Conclusions: FU induces mRNA levels of DPD, UP, and TS in cancer cell lines, but protein levels are reduced; the discrepancy between mRNA and protein levels following FU treatment underscores the need for evaluation of colon cancer samples to assess response to FU therapy. This work was supported by a Supplement of the MSM/TU/UABCCC U54 Partnership grant (3U54-CA118948-09S1) from NIH. Citation Format: Esther A. Suswam, Gaurav Kumar, Hyung-Gyoon Kim, Mohamed Osmar, Mahmoud H. el Kouni, Upender Manne. In vitro characterization of PTAU and FU interactions in colon cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2159.

Abstract A54: Inhibition of FOXM1 by thiostrepton increases sensitivity to 5-fluorouracil (5-FU) by downregulating thymidylate synthase (TS) in colorectal cancer

Abstract Background: 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapy drugs for the treatment of colorectal cancer in the adjuvant and metastatic disease settings. One of the main targets of 5-FU is the enzyme thymidylate synthase (TS). High expression levels of TS in colorectal tumors removed from patients have been associated with drug resistance. The Forkhead box transcription factor M1 (FOXM1) has been demonstrated to regulate several aspects in cancer cells including: cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal, and has been implicated in drug resistance in breast cancer. Thiostrepton is a thiazole antibiotic which selectively inhibits FOXM1. The relationship between FOXM1/TS and 5-FU resistance has not previously been established and may present a new treatment strategy. Purpose: In this study we sought to determine 1) the role of FOXM1 in regulation of TS, 2) whether inhibition of FOXM1 using thiostrepton or transient knockdown using a small interfering RNA would inhibit the TS expression in colorectal cancer cells, 3) whether combined therapy with 5-FU and thiostrepton would have a synergistic effect on cell survival, and 4) the effect of treatment with thiostrepton/ 5-FU/ combined thiostrepton and 5-FU on cellular migration and colony formation. Methods and results: HCT116, DLD1, and HT29 cells were treated with 0.5ug/ml 5-FU. TS and FOXM1 protein and mRNA expression were determined using Western blot and RTPCR. 5-FU treatment resulted in an initial increase (6h) followed by a decrease (48h) of both TS and FOXM1 in p53 wild type HCT116 cells, in p53 mutant DLD1 and HT29 cells whilst an initial increase was observed in both FOXM1 and TS there was no subsequent decrease. SRB assay was used to quantify the IC50 of 5-FU in HCT116, DLD1 and HT29 and was found to be 0.67±.23, 0.68±.22 and 3.09±.39 respectively. Transient knockdown of FOXM1 led to a significant decrease of TS in mRNA but not protein in HCT116 cells. For thiostrepton the IC50 was found to be 0.60uM, 1.07uM, and 3.54uM, in HCT116, DLD1 and HT29 cells, respectively. Combination of 5-FU and thiostrepton showed synergistic effects in all 3 colon cancer cell lines. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cell lines to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). 3 negative primers were designed further to 2000bp upstream region. ChIP data revealed enrichment for FOXM1 at the TS promoter (0-1500 bp upstream) and no enrichment observed in the negative control. Inhibition of FOXM1 by thiostrepton led to a drop in enrichment in the TS promoter region. These results confirmed the role of FOXM1 in TS regulation. Chip Sequencing (ChIP-seq) was then performed to study further FOXM1 targets and binding sites of FOXM1 to other well-known 5-FU targets such as thymidine phosphorylase (TP) and thymidine kinase 1 (TK-1). The FOXM1 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. ChIP-seq confirmed that FOXM1 significantly binds to both these factors. FOXM1 binding to cell cycle regulatory genes E2F2 and E2F3 was also shown in both cell lines. Combination treatment of 5-FU and thiostrepton significantly decreases colony formation in HCT116, DLD1 and HT29 cell lines. 5-FU and thiostrepton combination also decreases migration in HCT116 cells. Conclusion: FOXM1 regulates the 5-FU target gene TS, and inhibition of FOXM1 acts enhances the cytotoxic effect of 5-FU in colorectal cancer cells and warrants further investigation as a new treatment strategy. Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Lam W. Eric, Laura Kenny. Inhibition of FOXM1 by thiostrepton increases sensitivity to 5-fluorouracil (5-FU) by downregulating thymidylate synthase (TS) in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A54.

Prognostic and predictive significance of thymidylate synthase protein expression in non-small cell lung cancer: a systematic review and meta-analysis.

It remains controversial whether thymidylate synthase (TS) protein expression is associated with survival for patients with non-small cell lung cancer (NSCLC). To evaluate prognostic and predictive significance of tumor TS protein level in NSCLC. Electronic searches were performed for relevant studies in PubMed, EMBASE, Web of Science, and Chinese Biomedical Literature Database. Hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS) were pooled for meta-analysis. Subgroup and sensitivity analyses were performed. Publication bias was evaluated by funnel plot and Begg's test. Twenty-four studies, including 2280 patients, were eligible. This analysis showed that patients with low TS expression had statistically significantly longer OS and PFS than those with high TS (HR=0.51 and HR=0.49, respectively). Based on TS-targeted drug use status, TS expression was significantly associated with OS in pemetrexed (HR=0.42) and 5-Fluorouracil subgroups (HR=0.34), but not in no TS-targeted drug subgroup. There were similar results for PFS analyses. Sensitivity analysis indicated that the results were robust. Begg's test did not reveal any publication bias. Low TS protein expression is a favorable predictive factor for better OS/PFS in NSCLC patients treated with TS-targeted drugs. Prognostic value of TS protein expression needs further validation.

Abstract 1684: Histone deacetylase inhibition enhances Pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer models

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM's molecular targets) expression. Histone deacetylase inhibitors (HDACi) modulate the expression of multiple genes, including thymidylate synthase (TS), one of the main targets of Pemetrexed. Since high levels of TS have been linked to clinical resistance to Pemetrexed in non-small cell lung cancer (NSCLC), we investigated the molecular and functional effects of combined Pemetrexed and ITF2357, a class I/II HDACi currently in clinical trials as anti-cancer agents .ITF2357 reduced TS mRNA and protein expression, potentially affecting sensitivity to Pemetrexed. When the two drugs were used in combiation, their sequence of administration was critical in determining the pharmacologic response: in both NSCLC cell lines and LCSC, a strikingly synergistic reduction in cell viability was observed with the sequence Pemetrexed followed by ITF2357, in both adenocarcinoma and squamous cell carcinoma models. Cell death induced by the combination involved both apoptosis and autophagy. Genetic and pharmacological approaches provided an interesting link between these two pathways and showed that sequential Pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential Pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival. Overall, this data provide a strong rationale for the clinical development of sequential schedules employing Pemetrexed followed by HDAC inhibition in NSCLC. Citation Format: Daniela Trisciuoglio, Marianna Desideri, Teresa De Luca, Marta Di Martile, Chiara Gabellini, Adriana Eramo, Ruggero De Maria, Michele Milella, Donatella Del Bufalo. Histone deacetylase inhibition enhances Pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1684. doi:10.1158/1538-7445.AM2014-1684

Correlation between expression of thymidylate synthase and clinical outcome of advanced gastric cancer treated with capecitabine alone chemotherapy.

Thymidylate synthase (TS) is a prognostic marker in various tumors. However, the results of previous investigations in gastric cancer (GC) were controversial. The objective of this article is to investigate whether TS expression is associated with clinical outcome in advanced GC receiving capecitabine alone chemotherapy. The study reviewed 58 cases of advanced GC in patients aged ≥65years between December 2008 and June 2012. All patients were treated with capecitabine alone chemotherapy. Immunohistochemical staining for TS protein expression was performed. The relationships between TS expression and clinicopathological characteristics (included age, gender, number of metastatic sites, Eastern Cooperative Oncology Group (ECOG) score, differentiation, and lymph node metastatic status), chemotherapy response, progression-free survival (PFS), and overall survival (OS) were evaluated. There was no association between TS expression and age, gender, number of metastatic sites, ECOG score, differentiation, and lymph node metastatic status (P > 0.05). The chemotherapy response rates among patients with low- and high-level expression of TS protein were 52.0% (13/25) and 21.2% (7/33), respectively (χ (2) = 5.968, P = 0.015). The median PFS and OS in patients with low-level TS expression were significantly longer than those with high-level TS expression (PFS 8.0 vs 2.8m, P = 0.001; OS 13.3 vs 7.9m, P = 0.002, respectively). Multivariate Cox regression analysis revealed that TS expression was independent risk factor for OS (hazard ratio (HR) 0.237; 95% confidence interval (CI) 0.108 to 0.520; P = 0.000). The present study demonstrates that TS expression is associated with chemotherapy response, PFS, and OS in advanced GC patients treated with capecitabine alone chemotherapy.

192P - Prognostic Biomarkers in Malignant Pleural Mesothelioma

ABSTRACT Aim: The standard of care for malignant pleural mesothelioma (MPM) includes combination of platinum compounds and pemetrexed. Despite this, the median survival of MPM patients is still bad (12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), a folic enzyme also involved in ex novo synthesis of DNA. This study aims to analyse retrospectively, in a large series of MPM specimens, the ERCC1 and TS gene and protein expression to investigate their prognostic and predictive role. Methods: Pleural biopsies were collected from140 consecutive MPM patients (98 males and 42 females, mean age 68 years, range 27-90). Histologic subtypes were: epithelioid (#103, 73%), sarcomatoid (#19, 14%) and biphasic (#18, 13%). At the end of the study 113 patients were died (average disease specific survival (DSS) 13 months; range 1-60); mRNA was extracted from formalin fixed paraffin embedded blocks and reverse transcribed in cDNA to perform gene expression assay by qRT-PCR. Immunohistochemistry was performed by anti-ERCC1 and anti-TS mAbs and the results were scored from 0 to 3 depending on the percentage of positive tumour cells and staining intensity. The results were statistically correlated with the clinical features and with the DSS of the patients respectively by Fisher's exact test and Logrank test. Results: Of 140 patients 102 received platinum and pemetrexed (in single or in combination), the remaining were untreated due to bad performance status, advanced age or refusing therapy. Statistically significant correlation was found between the lack of protein expression of ERCC1 and DSS in the whole cohort (P = .03). Low levels of TS mRNA were significantly correlated with the DSS in the whole cohort (P = .01) and in the untreated subgroup (including patients not receiving pemetrexed) (P = .04), whereas did not in the subgroup of pemetrexed treated patients Conclusions: ERCC1 protein expression, scored by IHC, and TS gene expression, evaluated by mRNA analysis, seem both important prognostic markers; we suggest their evaluation in routine biopsies from patients with MPM. Disclosure: All authors have declared no conflicts of interest.