Thymidylate Synthase mRNA Research Articles

A practical approach for assessing chemosensitivity in colorectal cancer cell lines by comparative analysis of cell viability and thymidylate synthase mRNA expression

PurposeThe purpose of this study is to suggest a probable problem in chemosensitivity tests performed in practice and to speculate on practicable measures for more accurate chemosensitivity evaluation.MethodsThree colorectal cancer cells (RSC, RRC1, and RRC2) were treated with 5-fluorouracil (5-FU). Inhibition percentage (%inhibition) of cancer cells and relative quantitation of thymidylate synthase (TS) mRNA were measured on day 2, day 5 after replacement of 70% media on day 2, day 7, and day 3 after replacement of all media on day 7. Doses that produced 50% inhibition (Dm) were calculated to evaluate drug effect. Relative quantitation of TS mRNA and correlations between TS mRNA levels and 5-FU concentrations were analyzed.ResultsRRC1 was more resistant than RRC2 on day 7, but Dm value of RRC2 increased three days after replacement of media from 12.3 to 18.1. Mean TS mRNA levels of RSC on D2 and D7 were significantly lower than those of RRC1 and RRC2, respectively (P = 0.004, P = 0.004 on D2; P = 0.010, P = 0.006 on D7). TS mRNA levels in RRC1 were significantly reversely correlated with 5-FU concentrations on day 2 (correlation coefficient = -0.867, P = 0.015). On the other hand, correlations were not significant in RRC2 (r = 0.067).ConclusionEvaluating %inhibition of cancer cells at one point in chemosensitivity tests seems to be inadequate in determining chemotherapeutic regimens. Multilateral approaches, such as trials evaluating cancer cell survival before and after media replacement and correlations between TS mRNA levels and 5-FU concentrations, needs to be implemented for the practical application of chemosensitivity tests.

Role of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA in intrahepatic cholangiocarcinoma

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are important enzymes in the metabolism of 5-fluorouracil and possible predictive markers. We conducted this study to clarify if TS and DPD gene expressions are a prognostic indicator for intrahepatic cholangiocarcinoma (IHCC). The subjects of this study were 21 patients with IHCC who had undergone surgical resection. Intratumoral TS and DPD mRNA expressions were examined using the Danenberg tumor profile method and classified into two groups according to the median value of each. We then compared the clinicopathological variables, including prognosis, between the high and low expression groups. Low DPD expression was correlated with macroscopic type, namely, mass-forming + infiltrative (P = 0.08). Postoperative survival rates in the low DPD expression group were significantly lower than those in the high DPD expression group. Multivariate analysis revealed macroscopic type to be an independent prognostic factor, whereas TS mRNA expression did not correlate with any clinicopathological variables, including prognosis. Low DPD mRNA expression was related to macroscopic type and associated with poor prognosis. DPD mRNA expression in tumor cells is suggested to be an important regulator of malignant behavior in IHCC.

Response prediction in metastasised colorectal cancer using intratumoural thymidylate synthase: Results of a randomised multicentre trial

BackgroundMolecular markers to predict response to 5-fluorouracil (FU)-based treatment of recurrent or metastasised colorectal cancer (mCRC) are not established. The aim of this trial was to determine the value of thymidylate synthase (TS), a key enzyme of DNA synthesis and target of 5-FU, to predict response to chemotherapy of mCRC. MethodsTumour tissue was obtained from 168 patients with mCRC for relative thymidylate synthase (TS) mRNA quantitation. Patients were randomised to receive either 5-FU/folinic acid (FA, FUFA) alone or in combination with irinotecan 5-fluorouracil/folinic acid and irinotecan (FOLFIRI) stratified by TS (low versus high). Primary end-point was overall response to first-line treatment among TS high patients. All parties, except for the randomisation centre, were blinded for TS status. ResultsBiopsies (n=168) were taken without complications. TS levels were available for 147 patients (87.5%). Analysing response to FUFA and FOLFIRI in the per protocol set (n=119) after un-blinding TS in the data base revealed a trend to better overall response to FOLFIRI (9/19, 47%) in TS high compared to FUFA (5/23, 22%, p=0.077). In patients with biopsies taken from liver lesions (n=91) overall response to FOLFIRI and FUFA in TS high was 53% (9/17) and 18% (3/17), respectively (p=0.035). In patients with low TS, no remarkable difference in overall response to FOLFIRI and FUFA was observed. ConclusionsTaking a pre-treatment biopsy is a safe and feasible procedure in mCRC. After validation of our data in a larger group TS determination may have the potential to better help direct systemic treatment in patients with primarily non-resectable mCRC.

TS and DPD mRNA levels on formalin‐fixed paraffin‐embedded specimens as predictors for distant recurrence of rectal cancer treated with preoperative chemoradiotherapy

Distant metastatic relapse occurs in approximately 20% of rectal cancer patients treated with 5-fluorouracil (5-FU)-based chemoradiotherapy (CRT) followed by surgery. This study aimed to investigate mRNA level of 5-FU metabolizing enzymes in post-treatment specimens and to evaluate their predictive value of distant recurrence after CRT. Forty patients with rectal cancer underwent 5-FU-based CRT followed by surgery. After microdissection, total RNA of residual cancer was isolated from formalin-fixed paraffin-embedded (FFPE) specimens. Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT) as 5-FU metabolizing enzyme were measured using real-time reverse transcription polymerase chain reaction. Patients (n = 6) who developed distant recurrence had a significantly higher TS (P = 0.01), DPD (P = 0.02), and TP (P = 0.01) levels, compared with those patients (n = 34) without recurrence. High TS, DPD, and positive lymph node metastasis (pN) were significantly poorer prognostic factors for DFS (TS: P < 0.01, DPD: P < 0.01, pN: P < 0.05). High TS and DPD mRNA levels on FFPE specimens may predict distant recurrence of rectal cancer treated with 5-FU-based preoperative CRT followed by surgery. Expression analysis of 5-FU metabolizing enzyme in residual cancer may be useful for treatment stratification and clinical management in these patients.

Reexamining a proposal

The DNA replicative gene, thymidylate synthase (TYMS), is inhibited upon treatment with the anticancer drug 5-fluorouracil (5FU). TYMS has 28-bp tandem repeat sequences or VNTR (variable numbers of tandem repeats) in the 5'-untranslated region (5'-UTR). The number of these repeats is variable in any given population, but the most prevalent are double (2R) and triple (3R) repeat sequences. A single G/C nucleotide polymorphism in the triple repeat sequence gives rise to a 3Rc or a 3Rg triple repeat structure. A widely cited literature used plasmid constructs of the 5'-UTR and proposed that genotyping the TYMS UTRs would predict the efficiency of Tyms protein translation, justifying altered therapeutic dosage of 5FU. Prior studies had unusual features in experimental design, such as using the firefly Kozak sequence in place of the native human TYMS Kozak sequence to determine the ribosomal translational efficiency of TYMS mRNA. Our results using transient transfection, antibiotic-selected pools of transfected cells, and stably transfected clones, while using plasmids having native human Kozak sequence, refute the earlier results.

The presence of three repeats in the 5' UTR region of thymidylate synthase (TS) is associated with increased TS mRNA expression in cultured human cancer cell lines in vitro

Thymidylate synthase (TS) gene contains 28-bp polymorphic sequence and 6-bp insert at the 5'- and 3'-untranslated region, respectively. We investigated the role of these two polymorphic traits on TS mRNA expression in nine different cultured human cancer cell lines in vitro. Three cell lines each were 2R/2R, 2R/3R and 3R/3R genotypes. Six of the nine cell lines tested homozygous for the presence of 6-bp insert (+6/+6) and the rest three lacked this insert (-6/-6). TS expression analyses associated homozygous three repeats (3R/3R) to higher TS expression.

Anaplastic Lymphoma Kinase Translocation: A Predictive Biomarker of Pemetrexed in Patients with Non-small Cell Lung Cancer

This study compared the efficacy of pemetrexed in patients with anaplastic lymphoma kinase (ALK)-positive versus ALK-negative (epidermal growth factor receptor [EGFR] mutant or wild type [WT] for both ALK and EGFR) non-small cell lung cancer (NSCLC). Patients with advanced NSCLC who received second-line pemetrexed and beyond between March 2007 and April 2010 were screened for EGFR mutations and ALK rearrangements at Seoul National University Hospital. The clinical and in vitro efficacy of pemetrexed was evaluated for each genotypic group. Ninety-five NSCLC patients were genotyped as follows: 43 (45%) EGFR mutation, 15 (16%) ALK translocation, and 37 (39%) WT. The overall response rate was superior in ALK-translocated patients compared with EGFR mutant or WT patients (46.7 versus 4.7 versus 16.2%, p = 0.001). ALK-positive patients showed longer time to progression than EGFR mutant or WT patients (9.2 versus 1.4 versus 2.9 months, p = 0.001). ALK positivity alone was a significant predictor for overall response rate (hazard ratio [HR] = 0.07, 95% confidence interval [CI]: 0.01-0.32; p = 0.001) and time to progression (HR = 0.44, 95% CI: 0.24-0.80; p = 0.007). ALK positivity remained independently significant regardless of treatment line (HR = 0.43, 95% CI: 0.24-0.77; p = 0.005). Thymidylate synthase mRNA levels in ALK-positive cells were significantly lower compared with control cells (p < 0.05). Pemetrexed is an effective treatment in patients with ALK-positive NSCLC. ALK positivity was independently predictive of pemetrexed efficacy in NSCLC patients.

Significance of differential expression of thymidylate synthase in normal and primary tumor tissues from patients with colorectal cancer

The role of thymidylate synthase (TS) is essential as a key rate-limiting enzyme in DNA synthesis. It is the primary target of fluorouracil and its derivates in colorectal cancer. In this study, TS mRNA expression was examined in primary tumor and normal tissues from 76 patients with high- risk stage II/III colorectal cancer by laser capture microdissection and polymerase chain reaction. Thirty (39.47%) patients were found to have higher TS expression in primary tumors with earlier stage (P = 0.018), lower histological grades (P = 0.001) and high frequency microsatellite instability (P = 0.000). Multivariate analysis showed that microsatellite instability, histological grade and number of lymph nodes examined are independent prognostic markers.

TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials

To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

Thymidylate Synthase, Dihydropyrimidine Dehydrogenase, Thymidine Phosphorylase, Orotate Phosphoribosyltransferase mRNA Expression in Lung Cancer Metastatic Lymph Node Samples Obtained by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration – A Pilot Study

Introduction/BackgroundPyrimidine antimetabolites' target molecules thymidylate synthase (TS) and flopropyrimidine-metabolising enzymes such as dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyltransferase (OPRT) are known as biomarkers of 5-fluorouracil (5-FU) treatment. The purpose of this study was to evaluate the feasibility of mRNA expression analysis for TS, DPD, TP, and OPRT using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with non–small-cell lung cancer (NSCLC). Materials and MethodsMetastatic lymph nodes from 20 patients with NSCLC diagnosed by EBUS-TBNA were analyzed. After pathological diagnosis, additional sections were made and confirmed the existence of tumor cells by hematoxylin and eosin staining. Tumor cells were extracted using laser-captured microdissection and the mRNA levels were measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for each marker. ResultsThere were 17 mediastinal and 3 hilar lymph nodes. The histology included 13 cases of adenocarcinoma and 7 cases of squamous cell carcinoma. DPD and TP could be successfully analyzed in all samples and TS and OPRT could be measured in 18 samples. The median values of the relative TS, DPD, TP, and OPRT mRNA expression levels were 10.68, 6.23, 18.32, and 1.77, respectively. ConclusionTS, DPD, TP, and OPRT mRNA expression levels could be analyzed using metastatic lymph node samples obtained by EBUS-TBNA. The result of this pilot study may be applicable for further translational research for the treatment of locally advanced NSCLC.

The extract of Paris polyphylla exerts apoptotic induction and synergic antiproliferative effect with anticancer drugs in SMMC-7721 human liver cancer cells

Paris polyphylla is a famous traditional Chinese medicine with anti-inflammatory and hematischesis properties and has been shown recently to possess anticancer activities. However, the molecular mechanism underlying the anticancer effect of the herb is poorly understood. In this study, its ability to induce apoptosis and exert a synergistic antiproliferative effect by a combination with 5-fluorouracil (5-Fu) and Oxaliplatin (Oxa) were investigated in human SMMC-7721 liver cancer cells. Our findings have shown that the 95% ethanol extract of Paris polyphylla (PPE) inhibited the proliferation of SMMC-7721 cells dose- and time-dependently, as shown by loss of cell viability. The apoptotic induction was determined by Annexin-V/PI double staining assay, apoptotic body and swollen mitochondria observed under electron microscope, and breakdown of mitochondrial membrane potentials detected by JC-1 staining. Moreover, apoptosis in the SMMC-7721 cells was accompanied by S phase cell cycle arrest, the activation of pro-caspase 3, and a marked increase in Bax/Bcl-2 ratios due to upregulation of Bax and downregulation of Bcl-2 proteins expression by western blotting. In addition, we have observed that PPE increased the inhibitory effect of 5-Fu and Oxa on the proliferation of SMMC-7721 cells in vitro using Media-effect analysis. This combination regimen was more potent than either agent alone. Furthermore, the mRNA levels of thymidylate synthase (TS) and human excision repair cross-complementing (ERCC1) were suppressed when incubation with PPE, indicating that the effect of PPE on TS and ERCC1 mRNA levels at least partly caused the synergic effect. Overall, these results offer a potential mechanism for PPE-induced apoptosis and its synergic effect with anticarcinogenic drugs in SMMC-7721 cancer cells and indicate Paris polyphylla as a promising herb for chemoprevention and chemotherapy of hepotoma.

P109. Relative level of thymidylate synthase mRNA expression in primary tumors and normal tissues predicts survival of patients with oral tongue squamous cell carcinoma

P109. Relative level of thymidylate synthase mRNA expression in primary tumors and normal tissues predicts survival of patients with oral tongue squamous cell carcinoma

Combining Small Interfering RNAs Targeting Thymidylate Synthase and Thymidine Kinase 1 or 2 Sensitizes Human Tumor Cells to 5-Fluorodeoxyuridine and Pemetrexed

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein.

Abstract 5041: Thymidylate synthase (TS) in malignant pleural mesothelioma (MPM): Correlation with clinical outcome and pharmacological role in the synergistic interaction of vandetanib with pemetrexed/carboplatin combination

Abstract Pemetrexed improves the effect of platinum-compounds against MPM, but biomarkers of response and novel effective combinations are warranted. The present study evaluated the 1) correlation between expression and polymorphisms of pemetrexed's key target TS and outcome of MPM patients treated upfront with carboplatin/pemetrexed, and 2) pharmacological interaction of new targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, and enzastaurin) with carboplatin-pemetrexed in MPM cell lines. Analysis of TS TSER-2R/3R, mRNA and protein expression was performed by PCR and immunohistochemistry (using H-score) in tumors from 99 patients. The role of TS and other molecular determinants in the interaction of carboplatin, pemetrexed with new targeted compounds was investigated in the H2052, H2452, H28 and MSTO-211H cells. Drug interaction was studied using MTT and SRB assays and evaluated with combination index method. EGFR, Akt and Erk phosphorylation, as well as VEGF secretion, were analyzed with ELISA, whereas RT-PCR and western blot were performed to assess modulation of the expression of TS, E2F-1 and genes involved in DNA repair (ERCC1 and XPD). A significant correlation between low TS protein expression and response (odd ratio, 4.2; p=0.023), longer PFS (8vs6 months; hazard ratio [HR]:0.60: p=0.023), or OS (18vs9 months; HR:0.59; p=0.029) was found when patients were categorized according to median H-score. Similarly, patients with TS mRNA below the median had longer PFS and OS (p&amp;lt;0.001). The higher tertile of TS mRNA expression also correlated with higher risk of progressive disease (OR:2.5; p=0.044). At multivariate analysis, TS mRNA level and H-score confirmed their independent prognostic role for PFS and OS. No correlations were observed for TSER polymorphisms. Vandetanib emerged as the targeted compound with the most potent cell growth inhibitory effects, and interacted synergistically with carboplatin and pemetrexed in all cell lines, increasing apoptotic indices. Pemetrexed enhanced EGFR phosphorylation, but reduced Akt phosphorylation and ERCC1 and XPD expression. Conversely, vandetanib significantly downregulated EGFR, Erk and Akt phosphorylation, as well as E2F-1 and TS expression (eg, H28 cells had 4.8-fold TS mRNA decrease). In conclusion, low TS protein and mRNA levels were associated to response, longer PFS and OS. Furthermore, vandetanib improved pemetrexed-carboplatin activity against MPM cells, through modulation of critical molecular mechanisms, such as EGFR-Akt phosphorylation and TS expression. These data support further prospective trials for the validation of the prognostic/predictive role of TS in patients treated with pemetrexed-based regimens, as well as future studies on the integration of vandetanib in the treatment of MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5041. doi:10.1158/1538-7445.AM2011-5041

Abstract 3500: Histone deacetylator (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA) sensitizes squamous carcinoma cells of lung to pemetrexed

Abstract Objectives: The multitargeted antifolate pemetrexed is approved as a first-line or second-line treatment of nonsquamous nonsmall cell lung cancer. The expression of the thymidylate synthase (TS) gene may mediate resistance to pemetrexed. In this study, we investigated whether the pemetrexed in combination with SAHA enhances the cytotoxicity of squamous carcinoma cells of lung. Methods: Squamous carcinoma cells of lung (NCI-H157, HCC-95 and HCC-1588) were exposed to 10-3M to 10-7M of pemetrexed with various HDAC inhibitors and their cytotoxicities were analyzed using the colorimetric assay. Cell cycle and apoptosis analyses were evaluated by flow cytometry. The mRNA and cellular protein levels were detected by RT-PCR and western blot assay. Results: After NCI-H157 was exposed to SAHA, TS mRNA and protein expression levels were strongly suppressed by time and dose-dependent manner, respectively. Additionally, although pemetrexed activated phosphorylation of AMPK-α, secondary target (AICAR) of pemetrexed, co-treatment of pemetrexed and SAHA decreased phosphorylation of AMPK-α and mTOR pathway. NCI-H157, HCC-95, and HCC-1588 treated with sequential SAHA (24h) followed by SAHA plus pemetrexed (72h) represented more synergistic cytotoxicities than those treated with simultaneous 96h exposure combination (p&amp;lt;0.05). This sequential treatment was significantly associated with TS inhibition as well as apoptosis of squamous carcinoma cells. While pemetrexed increased expressions of p21, PARP, and cleaved caspase 3 and reduced expression of bcl-2, the combination of pemetrexed and SAHA additionally increased the pro-apoptotic molecules. Sequential treatment of SAHA followed by pemetrexed resulted in 10-fold and 1.6-fold higher sub-G1 portions than control and pemetrexed alone groups, respectively in NCI-H157 cell. In particular, the sequential administration produced more apoptotic index as compared to pemetrexed alone in NCI-H157 cell (27% vs 8%). Conclusion: Our results demonstrate SAHA reduces TS expression and sequential treatment with pemetrexed has a synergistic cytotoxicity in squamous cell carcinoma of lung. Sequential treatment of SAHA and pemetrexed might be a new strategy to overcome pemetrexed resistance in patients with squamous cell carcinoma of lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3500. doi:10.1158/1538-7445.AM2011-3500

Abstract 3514: Combination of 5-FU with ChoKα inhibitors as a new alternative therapy in colorectal cancer

Abstract Colorectal cancer (CRC) is the third major cause of cancer related deaths worldwide. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Despite the achieved improvements over the past years in cancer treatment, the design of novel targeted and combined therapies is still necessary. Choline kinase alpha (ChoKα), a phospholipid-related enzyme that plays a role in cell proliferation and transformation, has been found overexpressed in many different tumors, including colorectal tumors. ChoKα specific inhibitors, MN58b and TCD717, have potent antitumoral activity both in vitro and in vivo against several tumor-derived cell lines including CRC-derived tumour cells. TCD717 has recently entered clinical trials (http://clinicaltrials.gov/ct2/show/NCT01215864). We have evaluated the effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors. The sequential combination of ChoKα inhibitors with 5-FU resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. Key enzymes in the metabolic pathway of 5-FU such as Thymidylate synthase (TS) and Thymidine kinase (TK1) have been related to drug resistance, one of the major problems for the lack of success of 5-FU chemotherapy. TS is a key enzyme in the synthesis of DNA and the target enzyme of 5-FU. Several studies have demonstrated that the expression of TS mRNA or protein, predicts overall survival for colon cancer and correlates with resistance to 5-FU. Acquired resistance to 5-FU can also be caused by overproduction of TS that results from gene amplification. Regarding to 5-FU developed resistance, a salvaged pathway has been reported in which TK1 plays an important role. Thus, both TS and TK1 are directly involved in resistance to 5-FU therapy. To elucidate the mechanism of the observed synergistic effect, we examined the modulation of ChoKα inhibitors on the expression levels of TS and TK1. CRC cell lines treated with the sequential combination of ChoKα inhibitors and 5-FU show both mechanisms of TS inactivation, a formation of ternary complex due to 5-FU mechanism and a significant down-regulation of TS active due to ChoKα inhibitors, resulting in a total depletion of TS protein. We also observed that ChoKα inhibitors affects the levels of TK1, and provide the basis to support a new therapy focused on the combination of ChoKα inhibitors and 5-FU as a new approach for colorectal cancer patients. In addition, we suggest a promising role for ChoKα inhibitors as a new treatment for patients who had failed 5-FU chemotherapy or display high expression levels of TS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2011-3514

Abstract 1707: Establishment and characterization of pemetrexed-resistant lung cancer cell lines

Abstract Purpose: Pemetrexed (PEM), a multitargeted antifolate with manageable toxicity, is a most active drug against non-squamous non-small cell lung cancer (NSCLC); however, a majority of patients acquired the resistance to PEM eventually. To elucidate the mechanisms of acquired resistance to PEM, we established eight PEM-resistant NSCLC cell lines. Method: PC-9 and A549 cells (adenocarcinoma of the lung) were treated with step-wise increasing concentrations of PEM. Sublines, which were able to grow in the presence of low and high concentrations of PEM, were cloned with limiting dilutions and designated PC-9/PEML and PC-9/PEMH, respectively; A549/PEML and A549/PEMH, respectively. Growth inhibition was defined by MTT assay. Thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT) and multidrug resistance-associated protein 4 (MRP4) gene expressions and protein expressions were analyzed by quantitative reverse transcriptase PCR and immunoblotting. Result:Characteristics of PEM-resistant sublines are shown in the Table. All resistant sublines showed cross-resistant to cisplatin but not to docetaxel, vinorelbine, 5-fluorouracil and the active metabolite of irinotecan (SN-38). TS mRNA expression was significantly increased in all resistant sublines compared to the parent cells. TS mRNA expression was paralleled to the increment of TS protein. DHFR was significantly increased in the PEM-resistant A549 sublines. GARFT was not correlated with resistance to PEM. MRP4 was increased in PC-9/PEMH and A549/PEMH sublines. Conclusions: PEM-resistant NSCLC may be still sensitive to docetaxel, vinorelbine, 5-fluorouracil and irinotecan. High TS expression seems to be the primary resistant mechanism to PEM. MRP4 and DHFR expression might be elevated at higher resistance to PEM. Relative resistance (R.R.) was defined as the ratio of 50% inhibitory concentration (IC50) of resistant cells to IC50 of parent cells; *p&amp;lt;0.05; **p&amp;lt;0.01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1707. doi:10.1158/1538-7445.AM2011-1707

Thymidylate Synthase and Excision Repair Cross-Complementing Group-1 as Predictors of Responsiveness in Mesothelioma Patients Treated with Pemetrexed/Carboplatin

The pemetrexed/platinum agent combination represents the standard of care in first-line treatment for malignant pleural mesothelioma (MPM). However, there are no established indicators of responsiveness that can be used to optimize the treatment. This retrospective study aimed to assess the role of excision repair cross-complementing group-1 (ERCC1) and thymidylate synthase (TS) in tumors, and correlate expression levels and polymorphisms of these key determinants of drug activity with the outcome of MPM patients treated with carboplatin/pemetrexed in first-line setting. Analysis of TS and ERCC1 polymorphisms, mRNA and protein expression was done by PCR and immunohistochemistry [with the H-score (histologic score)] in tumor specimens from 126 MPM patients, including 99 carboplatin-/pemetrexed-treated patients. A significant correlation between low TS protein expression and disease control (DC) to carboplatin/pemetrexed therapy (P = 0.027), longer progression-free survival (PFS; P = 0.017), and longer overall survival (OS; P = 0.022) was found when patients were categorized according to median H-score. However, patients with the higher tertile of TS mRNA expression correlated with higher risk of developing progressive disease (P = 0.022), shorter PFS (P < 0.001), and shorter OS (P < 0.001). At multivariate analysis, the higher tertile of TS mRNA level and TS H-score confirmed their independent prognostic role for DC, PFS, and OS. No significant associations were found among ERCC1 protein expression, TS and ERCC1 polymorphisms, and clinical outcome. In our series of carboplatin-/pemetrexed-treated MPM patients, low TS protein and mRNA levels were significantly associated to DC, improved PFS, and OS. Prospective trials for the validation of the prognostic/predictive role of TS in MPM patients treated with pemetrexed-based regimens are warranted.

Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA.

A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-turn motif that also occurs in the `off' state of the human ribosomal decoding site RNA.

Analysis of 5-Fluorouracil–Related Enzymes in Pulmonary Neuroendocrine Carcinoma: Differences in Biological Properties Compared to Epithelial Carcinoma

BackgroundDihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidylate synthase (TS) levels correlate with sensitivity and resistance to 5-fluorouracil (5-FU). Few data are available on these enzymes in pulmonary neuroendocrine carcinoma, because 5-FU appears to have minimal effect on such carcinomas. Patients and MethodsThis study investigated 5-FU–related enzymes in large-cell neuroendocrine carcinoma (LCNEC; n = 31) and small-cell lung carcinoma (SCLC; n = 15), comparing expression levels with epithelial carcinomas including adenocarcinoma (ADC; n = 34) and squamous cell carcinoma (SCC; n = 13) obtained from 93 patients with primary lung tumors. Levels of 5-FU–related enzyme mRNA were analyzed by laser capture microdissection, compared with immunohistochemical findings and correlated with clinicopathologic factors. ResultsLCNEC and SCLC showed significantly higher TS and OPRT mRNA levels than ADC. SCLC exhibited significantly higher TS mRNA levels than LCNEC (P = .002). LCNEC displayed significantly lower DPD mRNA levels than ADC (P < .001), with a similar tendency in SCLC. SCC showed significantly lower DPD (P < .01) and higher OPRT (P < .001) mRNA levels than ADC. When we divide the data by pathology into epithelial carcinoma and neuroendocrine carcinoma, malignant potentials and prognoses correlated with mRNA levels in epithelial carcinoma, but not in neuroendocrine carcinoma. Immunohistochemically, neuroendocrine carcinomas were immunonegative for DPD. A significant correlation was found between enzymatic mRNA and protein expression for DPD (R = .500) and a weak correlation was observed for TS (R = .294). ConclusionNeuroendocrine carcinomas show characteristic patterns of expression for 5-FU–related enzymes, including low DPD mRNA and protein level and high TS mRNA level compared with adenocarcinomas. These results partially explain why 5-FU–based chemotherapy shows minimal efficacy against SCLC. Conversely, clinicopathological data and survival analysis indicates that 5-FU–related enzymes themselves might not affect the malignant potential of neuroendocrine carcinoma. Expressional differences in 5-FU–related enzymes among pathologies may provide valuable information for tailor-made chemotherapy.