Thymidylate Synthase Levels Research Articles

Inhibition of NF-κB translocation by curcumin analogs induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer

Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Curcumin is a potent inhibitor of NF-κB. EF31 and UBS109 are potent synthetic analogues of curcumin. We tested the hypothesis that inhibition of NF-κB translocation by curcumin and its analogs EF31 and UBS109 can inhibit cell cycle progression and downregulate TS levels in colorectal cancer (CRC) cell lines. Two CRC cell lines (HCT116 and HT-29) were either untreated (control) or treated with IC50 concentrations of curcumin, EF31 UBS109 led to G0/G1 cell cycle arrest. Treatment with curcumin, EF31 or UBS109 inhibited NF-κB, downregulated survival pathways and inhibited cell cycle progression. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. NF-κB over-expression induced E2F-1 and TS protein and mRNA levels in both cell lines. EF31 and UBS109 treatment significantly decreased tumor growth in compared to untreated tumors. EF31 and UBS109 are promising agents for the prevention and treatment of CRC.

Intrahepatic cholangiocarcinoma and gallbladder cancer: distinguishing molecular profiles to guide potential therapy

BackgroundChemotherapy regimens for intrahepatic cholangiocarcinoma (ICC) and gallbladder adenocarcinoma (GC) remain interchangeable; however, response rates are frequently suboptimal. Biomarkers from ICC and GC patients were interrogated to identify actionable differences with potential therapeutic implications. MethodsFrom 2009 to 2012, pathological specimens from 217 ICC and 28 GC patients referred to Caris Life Sciences were evaluated. Specific testing by immunohistochemical analysis for 17 different biomarkers was performed. ResultsIn the collective cohort (n= 245), actionable targets included: 95% low thymidylate synthase (TS), 82% low ribonucleotide reductase subunit M (RMM) 1 and 74% low excision repair cross complementation group (ERCC) 1, indicating potential susceptibility to fluoropyrimidines/capecitabine, gemcitabine and platinum agents, respectively. Additional targets included TOPO1 (53.3% high, Irinotecan), MGMT (50.3% low, temozolomide), TOP2A (33% high, anthracyclines) and PGP (30.1% low, taxanes). Subgroup analysis by tumour origin demonstrated a differential biomarker expression pattern with a higher frequency of ICC tumours showing low levels of TS (99% versus 72%, P < 0.01), and RRM1 (85% versus 64%, P = 0.02) when compared with GC. Conversely a greater frequency of GC demonstrated high levels of TOPO1 (76% versus 50%, P = 0.02) versus ICC, indicating a potential increased benefit from irinotecan. DiscussionDifferences in the molecular profiles between ICC and GC provide evidence that the two are distinct diseases, requiring different treatment strategies to optimize a response.

Thymidylate synthase expression as a predictive biomarker of pemetrexed sensitivity in advanced non-small cell lung cancer.

BackgroundAlthough it has been suggested that a high level of thymidylate synthase (TYMS) gene expression in malignant tumors is related to reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with the drug. The purpose of this study was to quantitatively assess the impact of TYMS gene expression in tumor cells as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer (NSCLC) treated at our institution.MethodsSixty-two NSCLC patients were included in this study: 16 patients received platins-pemetrexed as first-line NSCLC, and 46 pemetrexed in monotherapy as second- or subsequent-line treatment. Total mRNA was isolated and the expression of TYMS was analyzed by RT-qPCR. TYMS levels were calibrated against expression in normal lung tissue.ResultsTYMS overexpression was detected in 61 % of patients and low expression in 39 %. The response rate for patients with low TYMS expression was 0.29 compared with 0.03 in patients with overexpression (P = 0.025). A significant benefit was observed in patients with low expression both in time to progression (average TTP = 56 vs. 23 months, P = 0.001) and in overall survival (average OS = 60 vs. 25 months, P = 0.002).ConclusionsTYMS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced NSCLC patients.

Abstract 572: Proteomics analysis of the effect of fluorouracil (5FU) and 5FU/leucovorin (LV) on colorectal cancer (CRC) in patients

Abstract Background: 5FU is widely used and inhibits thymidylate synthase (TS); LV given in combination with 5FU enhances TS inhibition. TS levels and inhibition are associated with survival in advanced disease. The large variation in response between patients indicates that more mechanisms may be important. To make better treatment decisions, several additional biomarkers, next to TS, such as 5FU activation, breakdown and incorporation into RNA have been investigated. Several studies on global molecular profiling for 5FU activity have been performed on model systems and patients, showing that loss of 18p11-32, which harbors the TS gene, was associated with better survival. Aim: Identification of new potential mechanisms of action and potential biomarkers of 5FU with or without LV treatment using in-depth proteomics analysis of patient CRC tissues. Methods: Patients received approximately 48 hours before resection, a test dose of 5FU (500 mg/m2) (n = 5), 5FU with LV (500 mg/m2) (n = 7) or no test dose (n = 7). For proteomics analysis, samples of CRC resection material were lysed in SDS sample buffer, fractionated by 1D gel electrophoresis, followed by in-gel digestion and nano-liquid chromatography coupled to tandem mass spectrometry (QExactive). The MaxQuant tool was used for protein identification and quantification by spectral counting. Results: Our CRC proteome analysis identified a total of 6880 proteins and 874 with altered abundance (p&amp;lt; 0.05) upon treatments. Our results indicate that 5FU induces upregulation of proteins associated with the extracellular matrix, membrane vesicles, stress and immune responses and downregulates mitochondrial proteins, peroxisomes and (most profoundly) ribosomal proteins. Patients who received 5FU-LV also displayed upregulation of extracellular matrix and vesicle/granule proteins in addition to cell adhesion proteins, an extensive downregulation of ribosomal proteins and downregulation of mitochondrial and ubiquitin proteins. In addition, there is an upregulation of the GO-term ‘Extracellular region part’ in both 5FU and 5FU/LV treated patients, compared to controls. Top upregulated proteins were isoform NELF-D of negative elongation factor C (NELFCD), AMP deaminase 3 (AMPD) and myeloblastin (PRTN3), while top downregulated were neudesin (NENF), antigen KI-67 (MKI67) and HERC4. Conclusions: Proteome analysis revealed strong changes in the CRC proteome upon 48 hrs of 5FU +/- LV treatment, with regulated proteins involved in stress and immune responses, vesicular transport, protein synthesis and metabolism. A number of proteins were not yet associated to 5FU actions. These proteins when correlated in a larger cohort to clinical outcome may function as biomarkers for future treatment decision. Citation Format: Kees Smid, Erik Meijer, Thang V. Pham, Inge de Reus, Sander R. Piersma, Godefridus J. Peters, Connie R. Jimenez. Proteomics analysis of the effect of fluorouracil (5FU) and 5FU/leucovorin (LV) on colorectal cancer (CRC) in patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2015-572

Abstract 3778: Inhibition of NF-κB translocation induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer

Abstract Purpose Objectives: Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Curcumin is a potent inhibitor of NF-κB. We tested the hypothesis that NF-κB translocation inhibition by curcumin and its analogs can inhibit cell cycle progression and downregulate TS levels in colorectal cell lines. Methods: Two colorectal cancer cell lines were usedHCT-116 and HT-29. Cells were either untreated (control) or treated with curcumin (20μM), EF31 (7.5μM for HCT-116 and 2.5μM for HT-29) and UBS109 (620nM for HCT-116 and 1.25μM for HT-29) for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of curcumin and its analogues EF31 and UBS109 on different signal molecules involved in cell cycle arrest and DNA synthesis. Electromobility shift assay (EMSA) was performed in colon cancer cells to evaluate the effects of curcumin and its analogues on NF-κB activity. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of curcumin analogues. Results: Treatment with curcumin and its analogues inhibited cyclin D1, pRb and E2F-1 at the transcriptional and translational level and induced p21, leading to G0/G1 cell cycle arrest. NF-κB over-expression induced E2F-1 and TS protein and mRNA levels in both cell lines. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. Similar effects were observed in tumors from animals treated with EF31 and UBS109. Further, EF31 and UBS109 treatment significantly decreased the tumor growth compared to untreated tumors. Conclusion: In this study, we present in vitro and in vivo data to support the use of curcumin analogs in colorectal cancer based on their ability to reduce expression of proteins important for DNA repair and cell cycle progression leading to cell cycle arrest and tumor shrinkage. Accordingly, combining curcumin analogs with chemotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Ganji Purnachandra Nagaraju, Alese Olatunji, Sandhya Gupta, Mamoru Shoji, Bassel F. El-Rayes. Inhibition of NF-κB translocation induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2015-3778

Administration of adjuvant oral tegafur/uracil chemotherapy post hepatocellular carcinoma resection: A randomized controlled trial

Recurrence of hepatocellular carcinoma (HCC) after surgery is frequent, and is an important factor adversely influencing the long-term survival of patients. This prospective study evaluated whether adjuvant chemotherapy with oral tegafur/uracil (UFT) reduces the recurrence rate of HCC. In addition, expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) were investigated in resected tumors and nontumorous tissues, and the relationship between their expression and the effectiveness of UFT was examined. A total of 117 patients who underwent curative hepatic resection for HCC were randomly allocated to UFT 400mg/d (n=24, UFT group) or surgery alone (n=56, control group). The primary endpoint was the recurrence-free survival rate, and the secondary endpoint was the overall survival rate. Expression of the DPD and TS genes were quantified with TaqMan reverse transcription-polymerase chain reaction assay using β-actin as an internal standard. The cut-off value was set at the mean value of TS and DPD expression. Among the 61 patients in the UFT group, 37 patients (60.6%) discontinued UFT within 1 month. Recurrence-free survival (p=0.16) and overall survival (p=0.29) were similar in the two groups. In the UFT group, recurrence-free survival did not differ significantly between high-TS (TS>3.6) and high-DPD (DPD>8.9; n=10), and low-TS (TS≤3.6) and low-DPD (DPD≤8.9; n=9) groups. However, there was a significant difference between the two groups in overall survival (p=0.04). Peroral UFT administration fails to prolong the recurrence-free rates and overall survival rates, in comparison with surgery alone. However, oral administration of UFT may improve the survival of HCC patients when the levels of TS and DPD mRNA are low in the tumor tissue.

Tumor-related gene expression levels in pulmonary pleomorphic carcinoma.

BackgroundPulmonary pleomorphic carcinoma (PPC) is a rare type of non-small-cell lung cancer that belongs to the family of sarcomatoid carcinomas and is associated with poor prognosis. We investigated the expressions of tumor-related genes in resected PPC specimens.MethodsSpecimens resected from patients with PPC from July 2006 through April 2012 were investigated. Tumor segments were collected from the specimens by micro-dissection to extract mRNA, then RT-PCR was performed according to Dannenberg’s tumor profile method for semi-quantitation of tumor-related gene mRNA. To compare with other types of lung cancer, data from stage-matched adenocarcinoma (AC) and squamous cell carcinoma (SCC) cases in our database were also examined.ResultsThe gene expression levels of thymidylate synthase were significantly higher in PPC and SCC as compared to the AC specimens (p < 0.001). The levels of dihydropyrimidine dehydrogenase and thymidine phosphorylase mRNA in PPC showed a similar tendency to those in SCC, in contrast to AC. Furthermore, the expression level of excision repair cross-complementation group 1 mRNA in PPC specimens was similar to that reported in NSCLC, while the level of vascular endothelial growth factor (VEGF) expression was higher as compared to that reported for colorectal cancer.ConclusionsAlthough gene expression of tumor cannot be directly correlated to its sensitivity for anti-cancer drugs, it is likely that PPC tumors are not sensitive to anti-metabolic drugs. Anti-VEGF therapy may be a candidate for PPC, while cisplatin also remains a viable option.

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.

Expression of Ribonucleotide Reductase Subunit-2 and Thymidylate Synthase Correlates with Poor Prognosis in Patients with Resected Stages I-III Non-Small Cell Lung Cancer.

Biomarkers can help to identify patients with early-stages or locally advanced non-small cell lung cancer (NSCLC) who have high risk of relapse and poor prognosis. To correlate the expression of seven biomarkers involved in DNA synthesis and repair and in cell division with clinical outcome, we consecutively collected 82 tumour tissues from radically resected NSCLC patients. The following biomarkers were investigated using IHC and qRT-PCR: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2), subunit p53R2, thymidylate synthase (TS), and class III beta-tubulin (TUBB3). Gene expression levels were also validated in an available NSCLC microarray dataset. Multivariate analysis identified the protein overexpression of RRM2 and TS as independent prognostic factors of shorter overall survival (OS). Kaplan-Meier analysis showed a trend in shorter OS for patients with RRM2, TS, and ERCC1, BRCA1 overexpressed tumours. For all of the biomarkers except TUBB3, the OS trends relative to the gene expression levels were in agreement with those relative to the protein expression levels. The NSCLC microarray dataset showed RRM2 and TS as biomarkers significantly associated with OS. This study suggests that high expression levels of RRM2 and TS might be negative prognostic factors for resected NSCLC patients.

Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location impacts the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the mRNA expression of proteins involved in major signaling pathways, including tumor growth (EGFR), angiogenesis (VEGFR2), DNA repair (ERCC1) and fluoropyrimidine metabolism (TS). FFPE tumor specimens from 431 advanced CRC patients were analyzed. The presence of 7 different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by RT-PCR. BRAF mutations were significantly more common in the proximal colon (p<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared to distal and proximal colon tumors (p=0.001), and increased TS levels compared to distal colon cancers (p=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR, and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

Abstract B05: Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model

Abstract Oncogenic KRAS is a key initiator in the development of pancreatic cancer; however, other aberrations are necessary for the progression into advanced metastatic disease. Our lab was the first to show that overexpression of Thymidylate Synthase (TS), an essential DNA synthesis and repair enzyme that is elevated in many common adult cancers, plays a direct role in promoting tumorigenesis. These findings suggest that TS is not a passive marker of proliferation, but rather an important oncogenic driver in cancer. To determine whether TS enhances the neoplastic effects of oncogenic KRAS, we established a novel animal model for pancreatic ductal adenocarcinoma (PDAC) by generating conditional KrasG12D mutant mice that express high levels of human TS (hTS) in the pancreas. We discovered that overexpression of hTS in the pancreas of Pdx1-cre-KrasG12D/+ mice significantly reduced survival of the hTS/KrasG12D/+ mice. Median survival of hTS/KrasG12D/+ mice was reduced by 50% as compared to KrasG12D/+ mice (112.5 vs 222 days, p=0.0005). To test whether TS accelerates the development of preneoplastic pancreatic ductal lesions and tumors, hTS/KrasG12D/+ and KrasG12D/+ mice were euthanized at 1 month intervals from 1.0 – 6.0 months of age and the percentage of ducts displaying normal, murine pancreatic intraepithelial neoplasia (mPanIN), or invasive PDAC was scored by a masked veterinary pathologist using world health organization (WHO) criteria. We found that hTS/KrasG12D/+ mice displayed a 10 fold increase in mPanIN2 and mPanIN3 lesions at 2 months of age as compared to KrasG12D/+ mice alone. At 3 months of age the hTS/KrasG12D/+ mice showed a 10 fold increase in PDAC formation as compared to KrasG12D/+ mice alone. To determine whether TS enhances the metastatic potential of oncogenic KRAS, lung and liver tissues were harvested from these mice and analyzed for the presence of metastasis. We found that hTS/KrasG12D/+ mice developed lung metastasis as early as 2 months of age, while KrasG12D/+ mice did not develop lung metastasis until 4 months of age. In addition, 60% of hTS/KrasG12D/+ mice in the 4 month age group displayed liver metastasis compared to 20% of KrasG12D/+ mice. Taken together, our data demonstrate that hTS overexpression in the pancreas 1) enhances the initiation of preneoplastic pancreatic ductal lesions, 2) accelerates pancreatic ductal adenocarcinoma development, 3) reduces survival, and 4) increases metastasis in hTS/KrasG12D/+ mice. These data demonstrate that hTS cooperates with oncogenic KRAS to accelerate the progression of pancreatic cancer. Therefore, we predict that combined inhibition of TS and KRAS signaling will maximize tumor regression. Our lab has identified 4 new TS allosteric inhibitors (provisional patent filed in 2013), which inhibit the catalytic activity of TS through a different binding site than 5-fluorouracil (5-FU). These lead allosteric inhibitors display mid-nanomolar to low micromolar activity in KRAS-mutant pancreatic cancer cell lines. Our goal is to combine these allosteric TS inhibitors with inhibitors of KRAS effectors, such as PI3K/AKT/mTOR and RAF/MEK/ERK pathways. Given the frequency of aberrant KRAS activation in pancreatic cancer and the striking effect of TS to enhance KRAS driven tumors in our hTS/KrasG12D/+ model, this combination strategy will be an important step in identifying novel treatments for pancreatic cancer. Citation Format: Rony A. Francois, Akbar Nawab, Min Chen, Mary K. Reinhard, Frederic J. Kaye, Maria Zajac-Kaye. Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B05. doi: 10.1158/1557-3125.RASONC14-B05

Predictive value of thymidylate synthase for the prognosis and survival of lung adenocarcinoma patients.

Chemotherapy represents an important treatment modality for lung adenocarcinoma. Thymidylate synthase (TS) is an essential enzyme in DNA synthesis, and its overexpression has been associated with reduced sensitivity to antifolate agents. The aim of the current study was to investigate the expression of TS and the effect on prognosis in lung adenocarcinoma patients. Adenocarcinoma and adjacent carcinoma tissues were resected from 100 patients with lung adenocarcinoma and the TS levels were detected by immunohistochemical analysis. The values for overall survival (OS) and disease-free survival (DFS) were determined using the Kaplan-Meier analysis. The results indicated that the TS protein was expressed predominantly in adenocarcinoma tissues, which exhibited higher TS expression compared with the adjacent tissues (P<0.001). The statistical analysis indicated that TS expression was associated with the clinical stage and history of smoking (P<0.05). The Kaplan-Meier analysis results indicated that the DFS and OS in patients with high TS expression levels were significantly shorter compared with those with low expression levels (P<0.05). In conclusion, the results from this study suggested that TS may serve as an independent predictive factor for survival rate, which may indicate the prognosis of lung adenocarcinoma patients.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: A prospective multi-institutional study in Japan

ObjectivesThis investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. Materials and methods236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2cm or greater), IB, and II tumors were given a dose of 250mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Results and conclusionAmong 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS.The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery.

Role of key TYMS polymorphisms on methotrexate therapeutic outcome in portuguese rheumatoid arthritis patients.

BackgroundTherapeutic outcome of rheumatoid arthritis (RA) patients treated with methotrexate (MTX) can be modulated by thymidylate synthase (TS) levels, which may be altered by genetic polymorphisms in TS gene (TYMS). This study aims to elucidate the influence of TYMS polymorphisms in MTX therapeutic outcome (regarding both clinical response and toxicity) in Portuguese RA patients.MethodsClinicopathological data from 233 Caucasian RA patients treated with MTX were collected, outcomes were defined and patients were genotyped for the following TYMS polymorphisms: 1) 28 base pairs (bp) variable number tandem repeat (rs34743033); 2) single nucleotide polymorphism C>G (rs2853542); and 3) 6 bp sequence deletion (1494del6, rs34489327). Chi-square and binary logistic regression analyses were performed, using genotype and haplotype-based approaches.ResultsConsidering TYMS genotypes, 3R3R (p = 0.005, OR = 2.34), 3RC3RG (p = 0.016, OR = 3.52) and 6bp− carriers (p = 0.011, OR = 1.96) were associated with non-response to MTX. Multivariate analysis confirmed the increased risk for non-response to MTX in 6bp− carriers (p = 0.016, OR = 2.74). Data demonstrated that TYMS polymorphisms were in linkage disequilibrium (p<0.00001). Haplotype multivariate analysis revealed that haplotypes harboring both 3R and 6bp− alleles were associated with non-response to MTX. Regarding MTX-related toxicity, no statistically significant differences were observed in relation to TYMS genotypes and haplotypes.ConclusionOur study reveals that TYMS polymorphisms could be important to help predicting clinical response to MTX in RA patients. Despite the potential of these findings, translation into clinical practice needs larger studies to confirm these evidences.

Abstract 3922: Deregulated thymidylate synthase promotes tumorigenecity in pancreatic neuroendocrine tumors

Abstract The incidence of pancreatic neuroendocrine tumors (PNET) is rapidly increasing and the prevalence in the US is now estimated to be over 100,000. The lack of a suitable animal model that recapitulates the human disease has limited development of new treatment for PNET. We established a new mouse model by taking advantage of our discovery that elevated levels of Thymidylate Synthase (TS) plays a direct causal role in tumorigenesis in vitro and that overexpression of human TS (hTS) in transgenic mice promotes development of adenomas in the endocrine pancreas. We crossed hTS transgenic mice with conditional MEN1 null mice that were previously shown to develop pancreatic islet carcinoma upon long latency. MEN1 was recently shown to be the most commonly mutated tumor suppressor gene in sporadic PNETs (44% of PNET patients have MEN1 mutations). Our newly established hTS/MEN1 mice model developed aggressive PNET with 100% penetrance due to overexpressed TS in MEN1 null mice. Transgenic overexpression of TS induced islet carcinoma with shortened latency as compared to MEN1-/- mice. The hTS/fMen1-/- mice develop islet carcinoma as early as 6 month of age whereas MEN1-/- mice develop islet carcinoma at 8 months of age. The median age of onset of islet carcinoma in hTS/MEN1-/- and MEN1-/- mice was 10.38±2.45 vs. 13.06±0.48 months; p&amp;lt;0.001 respectively. We also observed significant decrease of overall survival in hTS/MEN1-/-mice as compared to MEN1-/- mice (p&amp;lt;0.001). The median survival time of hTS/MEN1-/- mice was 10.73±0.22 months as compared to 13.07±0.58 months in MEN1-/- mice. In addition, overexpression of TS results in the increase of mutational frequency in tumors derived from the hTS/MEN1-/-as compared to MEN1-/- mice. We crossed hTS/MEN1-/- mice with Big Blue® transgenic mouse that serves as a mutation detection system. We analyzed mutation frequency in tumors isolated from several 10-12 months old hTS/MEN1-/- and MEN1-/- mice. We observed that overexpression of TS increase mutational frequency ranging from 4.38 x10E-5 to 8.39 x10E-5 in hTS/MEN1-/- as compared to control MEN1-/- mice (2.15 x10E-5 to 3.01 x10E-5). Sequencing analysis demonstrated that mutations such as transitions, transversions, insertions and deletions were 3.2 fold higher in tumors isolated from hTS/MEN1-/- as compared to control MEN1-/- mice. In summary, our results suggest that high levels of TS increase mutational frequency that may result in accelerated growth of islet cells tumors in hTS/MEN1 mice. This novel animal model will allow development of new strategies for targeting TS in combination with other mutational driven-events that are critical for the treatment and prevention of PNET. Citation Format: Kyungah Maeng, Hye Seung Lee, Min Chen, Maria Zajac-Kaye. Deregulated thymidylate synthase promotes tumorigenecity in pancreatic neuroendocrine tumors. [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 3922. doi:10.1158/1538-7445.AM2014-3922

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

Abstract CT310: Molecular correlates of activity and survival in a phase I/II trial of sorafenib plus gemcitabine and capecitabine for advanced renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is the most common malignancy of the kidneys. We conducted an NCI-sponsored New York Cancer Consortium phase I/II trial with the combination of sorafenib (SOR) plus the cytotoxic agents gemcitabine (GEM) and capecitabine (CAP).Thymidylate Synthase (TS) is the key enzyme in the catalysis of the methylation from dUMP to dTMP in the DNA synthetic process and high levels may denote poor prognosis, but higher sensitivity to 5-FU in primary cultured RCC cells. In proliferating cells, ribonucleotide reductase (RR) is a key enzyme of de novo nucleotide synthesis pathway; RRM1 expression predicts response to gemcitabine and prognosis in several solid tumors. Methods: 26 patients (pts) with advanced RCC (65% clear cell, 31% papillary; 23% sarcomatoid differentiation) and 0-2 lines of prior therapy were enrolled in the clinical trial (NCI 6981): 15 in the phase I dose-escalation portion which defined toxicity and recommended phase II dose (RP2D): SOR 200 mg BID D1-21,GEM 750 mg/m2 D1 &amp; D8, and CAP 415 mg/m2 BID D1-D14 in 21-day cycles and 11 pts in the phase II portion. Immunohistochemical staining of archival tissue was performed in 13 patients to determine TS and RRM1 status. Response rate (RR) was assessed by RECIST, association between response and marker expression by Fisher's exact test, and overall survival (OS) was calculated by Kaplan-Meier methodology with comparisons using log-rank. Results: Overall, in the phase I/II study (all doses), RR is 16%, progression free survival (PFS) 6.1 mo, OS 18.3 mo [95% CI 10.3, 26.4]. For pts treated at the RP2D, RR is 12.5%, PFS 5.0 mo, OS 18.3. Pts positive for TS (61.5%) or RRM1 (53.8%) expression tended to have higher likelihood of stable disease or better by RECIST (though statistical significance was not reached, likely because of small numbers with progressive disease). Interestingly, the OS analysis demonstrated that pts with TS tumor expression lived longer (57.5 vs 18.1 mo, p=0.002) as did those with TS expression in vasculature (72.5 vs 21.7 mo, p=0.003). RRM1 positive pts also tended to live longer (57.5 vs 18.1 mo, p=0.13). Conclusions: The combination of SOR, GEM, and CAP was tolerated at attenuated doses, but does not appear to have significantly better activity than historical controls. However, TS expression may predict response to capecitabine containing therapy in advanced RCC, which consistent with in-vitro data demonstrating higher sensitivity of cultured RCC cells that express higher TS levels to fluoropyrimidine therapy. These findings are hypothesis-generating and prospective validation is needed. Citation Format: Bishoy Faltas, Gurveen Kaur, Naveed Akhtar, Paul Christos, Brian Robinson, Beerinder Singh, Himisha Beltran, David Nanus, Scott T. Tagawa. Molecular correlates of activity and survival in a phase I/II trial of sorafenib plus gemcitabine and capecitabine for advanced renal cell carcinoma. [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 CT310. doi:10.1158/1538-7445.AM2014-CT310

Abstract 1308: Functional inhibition of HSP90 induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer

Abstract Purpose Objectives: Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Ganetespib is a potent functional inhibitor of HSP90 currently under evaluation in clinical trials. We tested the hypothesis that HSP90 functional inhibition by ganetespib can inhibit cell cycle progression and downregulate TS levels in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50 nM for 24 hours. Western blot and Real time-PCR analyses were carried out to determine the effect of ganetespib on different signal molecules involved in cell cycle arrest and DNA synthesis. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of ganetespib. Pre and post ganetespib treatment tumor biopsies from patients with rectal cancer on a phase I trial of ganetespib were analyzed using RT-PCR. Results: Treatment with ganetespib inhibited cyclin D1, pRb and E2F-1 at the transcriptional and translational level and induced p21, leading to G0/G1 cell cycle arrest. HSP90 knockdown inhibited E2F-1 and TS protein and mRNA levels in both cell lines. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. Similar effects were observed in tumors from animals treated with ganetespib. Further, ganetespib treatment significantly inhibited the tumor growth. Inhibition of cyclin D1, Rb, E2F-1, TS and upregulation of p21 at transcriptional levels were observed in post treatment rectal tumor biopsies obtained from patients receiving ganetespib. Conclusion: In this study, we present preclinical and clinical data to support the use of HSP90 inhibitors in colorectal cancer based on their ability to reduce expression of proteins important for DNA repair and cell cycle progression leading to cell cycle arrest and tumor shrinkage. Accordingly, combining HSP90 inhibitors with chemotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Ganji Purnachandra Nagaraju, Field F. Willingham, Kevin E. Woods, Patrick Sullivan, Jerome C. Landry, Roberto Diaz, Bassel F. El-Rayes. Functional inhibition of HSP90 induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer. [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 1308. doi:10.1158/1538-7445.AM2014-1308

HSP90 inhibition downregulates thymidylate synthase and sensitizes colorectal cancer cell lines to the effect of 5FU-based chemotherapy

Cell cycle progression and DNA synthesis are essential steps in cancer cell growth. Thymidylate synthase (TS) is a therapeutic target for 5FU. We tested the hypothesis that HSP90 transcriptional and functional inhibition can inhibit cell cycle progression, downregulate TS levels and sensitize colorectal cancer (CRC) cell lines to the effects of 5FU. Treatment with ganetespib (50 nM) for 24 hours inhibited cyclin D1 and pRb at the transcriptional and translational levels and induced p21, leading to G0/G1 cell cycle arrest in both CRC cell lines (HCT-116 and HT-29). This was associated with downregulation of E2F1 and its target gene TS. In addition, ganetespib inhibited PI3K/Akt and ERK signalling pathways. Similar effects were observed with HSP90 knockdown in both cell lines. Ganetespib sensitized CRC cell lines to the effects of oxaliplatin and 5FU. Similar effects were also observed in tumors from animals treated with ganetespib, oxaliplatin and 5FU. In this study, we present in vitro and animal data supporting that the targeting of HSP90 decreases CRC cell survival and proliferation. Ganetespib sensitizes CRC cell lines to the effects of 5FU-based chemotherapy. Combining HSP90 inhibitors with chemotherapy is a rational approach for future drug development in CRC.

584P - Low Level of Thymidylate Synthase Gene Expression in Tumor Tissues is Associated with Response to Preoperative Chemoradiotherapy Including S-1 or Uft in Patients with Rectal Cancer

ABSTRACT Aim: Preoperative chemoradiotherapy (CRT) including 5-fluorouracil-based oral drug, S-1 or uracil/tegafur (UFT), significantly decreases local recurrence in locally advanced rectal cancer. However, reliable biomarkers to predict the response remain to be established. In the present study, we examined the association of the response to CRT with the expression levels of 18 drug-related genes in tumor tissues. Methods: Eighty two patients with locally advanced rectal cancer who received preoperative CRT with UFT or S-1 (± bevacizumab) for 5 weeks were analyzed. We assessed pathological tumor response according to the Japanese classification of colorectal carcinoma criteria. A patient with Grade 2 or 3 was defined as a responder. The mRNA expressions of pyrimidine-related enzymes, reduced folate-related enzymes and radiation-related enzymes (18 genes) in tumor biopsy specimens before CRT were quantitatively evaluated using a RT-PCR assay. The relationships between tumor response and gene expression levels were analyzed. The patients were divided into low and high TYMS groups using the cut-off value determined by the receiver operating characteristic curve. Results: Pathological response was observed in 72% (59/82) of the patients. There was no significant difference in response rates among clinical parameters. The gene expression level of thymidylate synthase (TYMS) was significantly higher in nonresponders than in responders (p = 0.028). There were no significant associations of tumor response with the expressions of other genes such as folylpolyglutamate synthase (FPGS) and hypoxia inducible factor 1 alpha subunit (HIF1A). In high TYMS group (response rate; 48% (10/21)), the gene expression level of FPGS and HIF1A were higher in nonresponders than in responders (p = 0.035 and p = 0.036, respectively). Conclusions: The TYMS gene expression levels in tumor tissues may be useful for predicting the efficacy of preoperative CRT including S-1 or UFT in patients with rectal cancer. In the patients with rectal cancer having high expression of TYMS, combination chemotherapy with leucovorin may be required in order to potentiate TYMS inhibition of 5-FU-based drug. Disclosure: H. Nagase: Mr. H. Nagase is a emplloyee of Taiho Pharmaceutical Co.,Ltd. All other authors have declared no conflicts of interest.