High Thymidylate Synthase Levels Research Articles

Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors.

Cell division is driven by nucleic acid metabolism, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in nucleotide synthesis. As a result, thymidylate synthase has emerged as a critical target in chemotherapy. 5-Fluorouracil (5-FU) is currently being used to treat a wide range of cancers, including breast, pancreatic, head and neck, colorectal, ovarian, and gastric cancers The objective of this study was to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives (UD-1 to UD-5) and to evaluate their therapeutic potential in BC cells. One-pot organic synthesis processes using a single solvent were used for the synthesis of drug analogues of Uracil. Integrated bioinformatics using GEPIA2, UALCAN, and KM plotter were utilized to study the expression pattern and prognostic significance of TYMS, the key target gene of 5-fluorouracil in breast cancer patients. Cell viability, cell proliferation, and colony formation assays were used as in vitro methods to validate the in silico lead obtained. BC patients showed high levels of thymidylate synthase, and high expression of thymidylate synthase was found associated with poor prognosis. In silico studies indicated that synthesized uracil derivatives have a high affinity for thymidylate synthase. Notably, the uracil derivatives dramatically inhibited the proliferation and colonization potential of BC cells in vitro. In conclusion, our study identified novel uracil derivatives as promising therapeutic options for breast cancer patients expressing the augmented levels of thymidylate synthase.

Gastric cancer with microsatellite instability displays increased thymidylate synthase expression.

Gastric cancer (GC) with microsatellite instability (MSI) is a less aggressive disease and associated with resistance to 5-fluorouracil (5-FU)-based chemotherapy (CMT). Thymidylate synthase (TS) is inhibited by 5-FU, and another potential mediator of therapeutic resistance to 5-FU. Therefore, we aimed to analyze the association between MSI and TS expression in GC, and its impact on disease outcomes. We retrospectively evaluated GC who underwent D2-gastrectomy. MSI and TS were analyzed by immunohistochemistry. We also investigated p53 expression, PD-L1 status, and tumor-infiltrating lymphocytes (CD4and CD8). Out of 284 GC, 60 (21.1%) were MSI. Median TS-score for all cases was 16.5. TS expression was significantly higher in MSI compared to microsatellite-stable (MSS;p < 0.001). Considering both status, GC were classified in four groups: 167 (58.8%) MSS + TS-low; 57 (20.1%) MSS + TS-High; 24 (8.5%) MSI + TS-low; and 36 (12.7%) MSI + TS-high. MSI + TS-high group had less advanced pTNM stage, higher CD8+T cells levels (p < 0.001) and PD-L1 positivity (p < 0.001). Normal p53 expression was related to MSI GC (p < 0.001). Improved survival was observed in MSI + TS-high, but no survival benefit was seen with CMT. MSI GC was associated with high TS levels, which may explain therapeutic resistance to 5-FU. Additionally, MSI + TS-high showed better survival, but without improvement with CMT.

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.

Measurement of Nucleic Acid Metabolizing Enzymes in Stage III Colorectal Cancer Adds Precision to Adjuvant Fluorouracil and Leucovorin Therapy

1. Abstract 1.1. <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>Background <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>: Recurrence of colorectal cancer has been shown to be inhibited by adjuvant chemotherapy. To achieve effective adjuvant chemotherapy, a target molecule is necessary . Therefore, we performed a prospective clinical trial to determine the relationship between adjuvant chemotherapy outcomes and prognostic factors in colorectal cancer based on nucleic acid metabolizing enzyme levels. 1.2. Methods: A total of 147 patients with stage IIIA, IIIB, and IIIC cancer were selected for adjuvant chemotherapy. We analyzed Disease-Free Survival (DFS) as the primary endpoint and Overall Survival (OS) as the secondary endpoint in relation to the Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) levels as measured by enzyme-linked immunosorbent assay. According <span style=letter-spacing: -.15pt>to the levels of the mean preliminary TS of 21.0 ng/mg protein and DPD of 115 ng/mg protein, the patients were divided into 4 groups. They were then administered postoperative adjuvant chemotherapy that primarily included fluorouracil and Leucovorin (FL) or Uracil/Tegafur (UFT) and Leucovorin (UZEL) for 6 months. 1.3. Results: The 5-year DFS in advanced stage III cancer was 63.9%. A significant difference was observed between the LL (low TS and low DPD levels) and HL (high TS and low DPD levels) groups and the LH (low TS and high DPD levels) and HH (high TS and high DPD levels) groups. Multivariate<span style=letter-spacing: -.5pt> analysis revealed a significant difference<span style=letter-spacing: -.1pt> in stage, TS level, and TS and DPD levels. The OS was 79.6% for all the groups. A significant difference was observed between the LL, LH, and HL groups and the HH groups. Multivariate analysis revealed a significant difference<span style=letter-spacing: -.5pt> in the stage, age, TS level, and<span style=letter-spacing: -.55pt> TS and DPD levels. <span style=font-size:10.0pt;letter-spacing: -.15pt>1.4. Conclusions: Measurement of the TS and DPD levels of a primary colorectal cancer lesion as target molecules may be helpful for achieving an effective postoperative adjuvant chemotherapy. TS and DPD can potentially serve as more accurate predictors of OS than age and cancer stage. 2. Keywords: Adjuvant Chemotherapy; Colorectal Cancer; Metabolizing <span style=font-size: 10.0pt;mso-fareast-font-family: ms= mincho=> <span style=font-size: 10.0pt>Enzyme; Precision Medicine

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

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

Predictive Significance of Thymidylate Synthase Expression in Non-small Cell Lung Cancer.

To date, many studies have suggested that thymidylate synthase (TS) could be used as a prognostic and predictive marker in non-small cell lung cancer (NSCLC) patients. However, results have been contradictory. The aim of this study was to evaluate TS mRNA levels in tumor tissue of NSCLC patients who underwent complete surgical resection and to analyze its prognostic and predictive potential. The study group consisted of 64 patients who underwent curative lung resection. Paired lung tissue samples were taken directly from the tumor tissue and from adjacent, histologically cancer-free lung tissue. The quantitative estimation of TS expression was performed by reverse transcription real-time polymerase chain reaction (RT-qPCR). The relationship between TS expression level and disease-free interval (DFI) and overall survival (OS) was analyzed. There was significantly higher TS expression in NSCLC tumor tissue comparing to normal lung tissue. In the group of patients who received adjuvant chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine, we found shorter DFI (p=0.0473) and OS (p=0.0053) in those with high expression of TS. Our results demonstrated the relationship of high tumor tissue TS levels to adverse prognosis in patients undergoing adjuvant chemotherapy. TS is a non-specific tumor marker with respect to NSCLC, therefore we think that its best use would be as a member of the panel of predictors of adjuvant treatment efficacy.

Higher Tissue Levels of Thymidylate Synthase Determined by ELISA Are Associated with Poor Prognosis of Patients with Lung Cancer.

Thymidylate synthase (TS) is essential in thymidylate biosynthesis and DNA replication. Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in pyrimidine catabolism and is important in catabolism of 5-fluorouracil (5-FU). The significance of TS and DPD expressed in lung cancer remains controversial. Here we analyzed the relationship between TS and DPD expression and clinicopathological features of lung cancer. Enzyme-linked immunosorbent assays (ELISAs) were used to measure TS and DPD levels in paired tumor and non-tumor lung tissues obtained from 168 patients (107 adenocarcinomas, 39 squamous cell carcinomas, and 22 others), who had operations at the Shinshu University Hospital from 2004 to 2007 and were followed up for a median of 57.0 months. TS and DPD expression levels were higher in tumor tissues, and TS expression levels were significantly lower in adenocarcinomas than those in other subtypes. In addition, patients with low TS levels survived longer compared with patents with high TS levels. By contrast, DPD expression levels were not correlated with overall patient survival. Importantly, patients with low TS and DPD levels exhibited significantly prolonged survival than those with high TS and DPD. Among the 168 patients, 59 patients were treated with tegafur-uracil (UFT), a DPD-inhibitory fluoropyrimidine, and the UFT-treated patients with high TS and high DPD levels showed worst prognosis. Our study demonstrates a significant correlation between low TS expression levels and long-term prognosis of patients with lung cancer. Thus, ELISA is a clinically useful method to measure TS and DPD expression in lung cancer tissues.

A Study of Thymidylate Synthase Expression as a Biomarker for Resectable Colon Cancer: Alliance (Cancer and Leukemia Group B) 9581 and 89803.

Tumor levels of thymidylate synthase (TS), a target of 5-fluorouracil (5-FU)-based chemotherapy for colorectal cancer, have been studied as a predictive or prognostic biomarker with mixed results. Tumor TS levels were prospectively evaluated in two adjuvant therapy trials for patients with resected stage II or III colon cancer. TS expression was determined by standard immunohistochemistry and by automated quantitative analysis. Tumor mismatch repair deficiency (MMR-D) and BRAF c.1799T > A (p.V600E) mutation status were also examined. Relationships between tumor TS, MMR-D, and BRAF mutation status, overall survival (OS), and disease-free survival (DFS) were investigated in the subset of stage III patients. Patients whose tumors demonstrated high TS expression experienced better treatment outcomes, with DFS hazard ratio (HR) = 0.67, 95% confidence interval (CI) = 0.53, 0.84; and OS HR = 0.68, 95% CI = 0.53, 0.88, for high versus low TS expression, respectively. No significant interaction between TS expression and stage was observed (DFS: interaction HR = 0.94; OS: interaction HR = 0.94). Tumors with high TS expression were more likely to demonstrate MMR-D (22.2% vs. 12.8%; p = .0003). Patients whose tumors demonstrated both high TS and MMR-D had a 7-year DFS of 77%, compared with 58% for those whose tumors had low TS and were non-MMR-D (log-rank p = .0006). Tumor TS expression did not predict benefit of a particular therapeutic regimen. This large prospective analysis showed that high tumor TS levels were associated with improved DFS and OS following adjuvant therapy for colon cancer, although tumor TS expression did not predict benefit of 5-FU-based chemotherapy. The Oncologist 2017;22:107-114Implications for Practice: This study finds that measurement of tumor levels of thymidylate synthase is not helpful in assigning specific adjuvant treatment for colorectal cancer. It also highlights the importance of using prospective analyses within treatment clinical trials as the optimal method of determining biomarker utility.

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.

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

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

Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer.

BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.ResultsIn NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic 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.ConclusionsOverall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-230) contains supplementary material, which is available to authorized users.

Therapeutic enhancement of S‐1 with CPT‐11 through down‐regulation of thymidylate synthase in bladder cancer

Thymidylate synthase (TS), a target enzyme of 5-fluorouracil (5-FU), is significantly associated with prognosis in various cancers. Recently, it has been reported that S-1, a novel 5-FU-based agent has an effect on bladder cancer. However, in cells with high TS level, S-1 did not have significant effects. Therefore, we examined whether down-regulation of TS enhanced effects of S-1 in them. First, we measured TS level in an aggressive bladder cancer cell line, KU-19-19 by enzyme-linked immunosorbent assay (ELISA) and evaluated its sensitivity to 5-FU using a small interfering RNA (siRNA) for TS. Next, we measured TS mRNA after exposure to various agents. Finally, we evaluated enhancement of cytotoxicity of S-1 by CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) which down-regulated TS in in vivo study. The median TS and dihydropyrimidine dehydrogenase (DPD) level was 53.3 ng/mg and 80.3 ng/mg in KU-19-19 cells, respectively. The 5-FU treatment in KU-19-19 cells transfected with siRNA for TS gene (TYMS) inhibited cell growth more significantly than that for nontargeting control. Down-regulation of TS was observed after exposure to SN-38 (7-ethyl-10-hydroxycamptothecin) in a dose-dependent manner. The combination treatment of 5-FU and SN-38 significantly inhibited cell growth, as compared to the single treatment. Meanwhile, in cells transfected with siRNA for TYMS, neither an additive nor a synergistic effect was observed. Also, combined S-1 and CPT-11 dramatically inhibited tumor growth, compared to S-1 or CPT-11 alone in in vivo study. In conclusion, CPT-11 down-regulated TS level and enhanced the effect of S-1. Thus, the combination therapy with S-1 and CPT-11 might be a novel modality for bladder cancer, even with high TS level.This study confirmed that thymidylate synthase (TS) level in an aggressive human bladder cancer cell line, KU-19-19, was relatively higher than that in other cancer and presented that irinotecan (CPT-11) could down-regulate TS. Finally, the combination therapy with S-1 and CPT-11 resulted in significant tumor growth inhibition through down-regulation of TS in KU-19-19. Thus, combined S-1 and CPT-11 might be a novel treatment in bladder cancer, even with high TS.

Uncommon and parallel developmental patterns of thymidylate synthase expression and localization in Trichinella spiralis and Caenorhabditis elegans

Abstract Trichinella spiralis is a parasitic nematode causing trichinellosis, a serious disease, and Caenorhabditis elegans is a free-living nematode, which is used as a model in parasitological studies. High levels of thymidylate synthase (EC 2.1.1.45; ThyA) and certain other enzymes involved in thymidylate biosynthesis were found throughout T. spiralis and C. elegans developmental cycles, including developmentally arrested forms, that is, T. spiralis muscle larva and C. elegans dauer larva. As ThyA activity is characteristic for cells that left the G0 phase of the cell cycle, an exceptional regulation of the cell cycle in nematodes is suggested, manifested by a global cell cycle arrest in developmentally arrested larvae of the two species. ThyA immunolocalization during development of T. spiralis and C. elegans revealed the presence of high enzyme levels not only in the developing embryos, where it was expected, but also in gonad primordia, egg and sperm cells, nerve ring and secretory cells, opening to T. spiralis esophagus and C. elegans pharynx, where it may point to those cell populations remaining cell cycle arrested.

A phase 1 study of TRC102, an inhibitor of base excision repair, and pemetrexed in patients with advanced solid tumors

TRC102 potentiates the activity of cancer therapies that induce base excision repair (BER) including antimetabolite and alkylating agents. TRC102 rapidly and covalently binds to apurinic/apyrimidinic (AP) sites generated during BER, and TRC102-bound DNA causes topoisomerase II-dependent irreversible strand breaks and apoptosis. This study assessed the safety, maximum-tolerated dose (MTD), pharmacokinetics and pharmacodynamics of TRC102 alone and in combination with pemetrexed. Patients with advanced solid tumors received oral TRC102 daily for 4 days. Two weeks later, patients began standard-dose pemetrexed on day 1 in combination with oral TRC102 on days 1 to 4. The pemetrexed-TRC102 combination was repeated every 3 weeks until disease progression. Twenty-eight patients were treated with TRC102 at 15, 30, 60 or 100 mg/m(2)/d. The MTD was exceeded at 100 mg/m(2)/d due to grade 3 anemia in 50 % of patients. TRC102 exposure increased in proportion to dose with a mean t1/2 of 28 h. A pharmacodynamic assay confirmed that TRC102 binds to pemetrexed-induced AP sites at all doses studied. Stable disease or better was achieved in 15 of 25 patients evaluable for response (60 %), including one patient with recurrent metastatic oropharyngeal carcinoma that expressed high levels of thymidylate synthase, who achieved a partial response and was progression free for 14 months. When administered with pemetrexed, the maximum tolerated dose of oral TRC102 is 60 mg/m(2)/d for 4 days. Randomized controlled studies are planned to evaluate the clinical benefit of adding TRC102 to pemetrexed and other agents that induce BER.

Prognostic significance of 5-fluorouracil metabolism-relating enzymes and enhanced chemosensitivity to 5-fluorouracil by 5-chloro 2,4-dihydroxy-pyridine in urothelial carcinoma

BackgroundRecently, S-1, a novel 5-fluorouracil (5-FU)-based agent containing the strong dihydropyrimidine dehydrogenase (DPD) inhibitor, 5-chloro-2,4-dihydropyrimidine (CDHP) has been clinically used to treat various non-urothelial carcinomas (UC). High levels of thymidylate synthase (TS), the target enzyme of 5-FU and DPD which degrades the majority of 5-FU, are associated with poor prognosis in some cancers. However, only a few reports have dealt with this in UC. The aim of this study was to investigate the clinical significance of TS and DPD in upper tract urothelial carcinoma (UTUC) and evaluate the role of TS and DPD on the sensitivity of 5-FU in UC cell lines and the anti-tumor effect of S-1 in UC xenograft model.MethodsFirstly, we evaluated the immunohistochemical expression of TS and DPD in 176 patients with UTUC to determine their prognostic significance. Secondly, the levels of TS and DPD in UC cell lines were measured by ELISA and real-time PCR. Furthermore, the association between their levels and the sensitivity to 5-FU was examined using the small interfering RNA (siRNA) specific for TS and DPD. Thirdly, the anti-tumor effect of S-1 was evaluated in UC xenograft model.ResultsImmunohistochemical evaluation of TS and DPD in UTUC human samples revealed that TS expression was significantly associated with stage, grade, and lymphovascular invasion and DPD expression was significantly associated with grade. Multivariate analysis revealed that high TS expression was an independent predictor of disease-specific survival in them. In in vitro study using UC cell lines, high levels of TS and DPD were associated with low response to 5-FU and these associations were confirmed with siRNA specific for TS and DPD. In in vivo study using UC xenograft model, S-1 treatment dramatically inhibited tumor growth compared to controls, tegafur, or UFT in UC tumor with a high level of DPD.ConclusionsTS plays an important role in the prognosis of UTUC and S-1 may be a key agent for UC tumor, especially with a high level of DPD.

Abstract 4514: A comparison of two methodologies to quantify Thymidylate synthase for predicting survival in patients with colorectal cancer treated on CALGB 89803

Abstract Background: Thymidylate synthase (TS) levels determined by immunohistochemistry (IHC) have been shown to be associated with decreased survival in retrospective studies of patients with colorectal cancer (CRC). The ratio of nuclear to cytoplasmic TS determined by automated quantitative analysis (AQUA™) has been reported to significantly predict a decreased time to recurrence in these same patients. The purpose of this study was to determine whether TS measured by the AQUA™ method predicts response in the CALGB 89803 patient sample, and to determine if the AQUA™ measurement of TS is prognostic compared to the standard IHC method. Methods: Tissue samples from 629 participants were evaluated from an IRB-approved randomized trial of 5-FU/LV/CPT-11 in patients with stage III CRC. TS levels were evaluated by IHC in 463 samples, by AQUA™ in 416 samples, and by both methods in 250 samples. TS staining by IHC was categorized as 0; 1+; 2+; 3+. TS staining by AQUA™ localizing to the nucleus, cytoplasm, and the ratio of the two (nucleus/cytoplasm), were classified as continuous measurements standardized by the highest observed value. The impact of each measure on survival (OS) and disease-free survival (DFS) was investigated using the logrank test and the proportional hazards model. Statistical correlations between the two measures were estimated using polyserial correlation. Results: In TS levels measured by IHC, there were no significant differences in OS. There was a significant difference in DFS (logrank p=0.047), with increased DFS among patients with higher TS levels. The nuclear and ratio AQUA™ TS measurements did not predict OS, but cytoplasmic TS marginally predicted OS (Cox model p=0.07). There were significant associations between nuclear and cytoplasmic TS and DFS (Cox model p=0.019 and p=0.007, respectively) with higher TS levels associated with increased DFS. DFS was marginally associated with the ratio (Cox model p=0.069). Correlation between TS staining measured by IHC and AQUA was low, while the correlation between the nuclear and cytoplasmic AQUA measurements was high (0.90 Pearson estimate). In multivariable models, the continuous measures of nuclear and cytoplasmic TS were each superior to TS measured by IHC in predicting DFS (logrank p=0.03 and 0.1 respectively); the ratio was not. Conclusions: TS levels measured by IHC and AQUA™ in the nucleus and cytoplasm predicted increased DFS with higher levels contrary to previous reports presenting significantly decreased survival for patients with higher TS tumor levels. The continuous measures for TS in the nucleus and cytoplasm were highly correlated and more strongly associated with outcome than TS measured by IHC. Given these results, further studies of TS as a prognostic predictor, and the use of the AQUA™ method as an alternate measurement of TS expression in colorectal tumors are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4514. doi:1538-7445.AM2012-4514

Expression Profiling-Based Subtyping Identifies Novel Non-small Cell Lung Cancer Subgroups and Implicates Putative Resistance to Pemetrexed Therapy

A challenge of cancer therapy is to optimize therapeutical options to individual patients. Cancers with similar histology may show dramatically different responses to therapy, indicating that a refined approach needs to be developed to classify tumors by intrinsic characteristics that may predict response to chemotherapy. Global expression profile-based classification has the potential to identify such tumor-intrinsic subclasses. Pemetrexed effectiveness has been related to the expression of its target thymidylate synthase. The relatively frequent resistance of squamous cell carcinoma to Pemetrexed is correlated with high levels of thymidylate synthase expression. A global expression profile-based molecular classification of non-small cell lung cancer (NSCLC) was performed. Gene expression was used to predict Pemetrexed responsiveness. The distinct molecular attributes of NSCLCs predicted likely to be resistant to Pemetrexed were bioinformatically characterized. We tested if routine immunohistochemical markers can be used to distinguish putative Pemetrexed responders, predicted by gene signatures, from nonresponders. Ninety NSCLCs were divided into six subclasses by gene expression signatures. The relevance of this novel phenotyping was linked to other tumor characteristics. Two of the subclasses correlated to putative Pemetrexed resistance. In addition, the identified signature genes characterizing putative Pemetrexed responsiveness predicted therapeutic benefit in a subset of squamous cell carcinoma. Gene expression signatures can be used to identify NSCLC subgroups and have potential to predict resistance to Pemetrexed therapy. We suggest that a combination of classical pathological markers can be used to identify molecular tumor subclasses associated with predicted Pemetrexed response.