Thymidylate Synthase Overexpression Research Articles

Enhanced anticancer effect of thymidylate synthase dimer disrupters by promoting intracellular accumulation.

Thymidylate synthase (TS) plays a crucial role in cellular growth, proliferation, DNA synthesis, and repair, thus gaining attention for targeted therapies in cancer. TS overexpression and the altered pharmacokinetics of anti-TS drugs are among the most prominent causes of cellular resistance. Decreased drug influx and/or efficient efflux result in reduced drug access to the intracellular targets. In this study, we have evaluated and demonstrated the increased cytotoxic efficacy of novel TS dimer disrupters (Ddis) in the presence of specific inhibitors of drug efflux protein pumps in ovarian and colon cancer cells, suggesting that these compounds are substrates of the cellular drug extruders. A second strategy adopted to favor intracellular accumulation was to employ, as a drug delivery system, a molecular tool able to help less lipophilic compounds to cross the cell membrane. The Ddis were delivered through the SAINT-Protein transfection agent. The observed cell-killing effects agreed with the reduction of TS protein level and cell cycle perturbation. Overall, this preclinical study suggests that the innovative TS dimer disrupters can be optimized by increasing their intracellular accumulation by both inhibiting their outflow and/or enhancing cellular uptake.

Abstract 2000: Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response

Abstract Rationale: Fluoropyrimidine drugs (FPs; e.g., 5-FU) are widely used to treat colorectal cancer (CRC), the 3rd leading cause of cancer-related mortality. However, acquired resistance to 5-FU is a principal cause of treatment failure in the metastatic setting. Further, previous studies selected for cells resistant to single agent 5-FU however 5-FU is invariably used clinically in combination with the reduced folate leucovorin (LV). Our studies focus on resistance mechanisms upon treatment with 5-FU+LV in folate-restricted media and test if CF10, a novel polymeric FP being developed in our laboratory displays activity in CRC cells resistant to 5-FU+LV. Experimental: We selected CRC cells (HCT-116, LS174T, HCT15, MC38) resistant to 5-FU+LV by selecting for survival upon culture in folate-restricted media with stepwise increased concentrations of 5-FU+LV over 5 months. Since elevated thymidylate synthase (TS) levels were observed in multiple studies selecting CRC cells for 5-FU resistance, we performed Western blot on cell lysates from parental and (5-FU+LV)-R cells. Since Myc and p53 expression are regulated in part via translational inhibition by TS binding to these mRNAs we also compared Myc and p53 levels in parental and (5-FU+LV)-R cells by Western blot. Further, since TS, Myc, and p53 protein stability are all regulated in part via O-GlcNAc conjugation we tested if OGT levels were changed in (5-FU+LV)-R cells. Resistance factors for CF10 were also determined in (5-FU+LV)-R cells. Results: A surprising result was that basal TS levels were reduced in (5-FU+LV)-R cells relative to the parental cells. We also observed that TS levels were increased in both parental and (5-FU+LV)-R cells by 5-FU+LV treatment however the effect was much greater in (5-FU+LV)-R cells. We observed strongly decreased p53 and Myc levels in the (5-FU+LV)-R cells. CF10 treatment further decreased p53 and Myc levels in parental cells, but increased p53 and further decreased Myc in (5-FU+LV)-R cells. In contrast, 5-FU alone treatment in (5-FU+LV)-R cells increased Myc and decreased p53 levels and further decreased in combination with LV. CF10 retained strong activity in (5-FU+LV)-R cells. Conclusions: Overall, we have validated that cancer cells can be cultured under conditions with human-like folate levels and selected for resistance to 5-FU+LV under folate-restricted conditions. The causes of 5-FU+LV resistance are multi-factorial and include TS overexpression induced by 5-FU+LV treatment that may be mediated by O-GlcNAc conjugation. We demonstrated that CF10 is more potent than 5-FU, and importantly its potency advantage is enhanced in cells selected for 5-FU+LV resistance. Our studies support CF10 translation for treatment of metastatic CRC in patients with progressive disease following treatment with combination therapy that includes 5-FU+LV. Citation Format: Charles C. Okechukwu, Xue Ma, William H. Gmeiner. Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2000.

An insight into thymidylate synthase inhibitor as anticancer agents: an explicative review.

Cancer, a widespread challenge to global health, remains a puzzle of intricate molecular dynamics. This review article delves into the mystery of cancer, with a keen focus on understanding the contributory role of thymidylate synthase (TS) in cancer. TS, a vital enzyme in DNA synthesis and repair, emerges as a significant player in the narrative of cancer development. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is a major step in producing DNA. Numerous malignancies, including those of the breast, colon, lung, and ovary, have been linked to dysregulation of TS activity. Overexpression or mutations of TS lead to uncontrolled cell proliferation and tumorigenesis molecular interactions and signalling pathways involving TS come under scrutiny, revealing the nuanced connections that propel its involvement in cancer progression. Beyond overexpression and mutations, there emerges a subtle layer of regulation that involves microRNAs (miRNAs). These tiny particles attach to the TS messenger RNA, causing translational repression or its degradation, which in turn affects TS activity. Moving towards the therapeutic realm, thymidylate synthase inhibition acts as a promising anti-cancer strategy. Targeting TS with small-molecule inhibitors could provide a novel approach to treat various cancers. By reducing the number of available nucleotides, TS inhibition would slow down or halt cancer cell division, thus depriving the tumor of the building blocks required for its proliferation and growth. The aim is to assess the viability and effectiveness of targeting TS to halt or slow down cancer progression. There is growing evidence that, in comparison to traditional TS inhibitors, few novel antifolate TS inhibitors are effective against a wider variety of neoplasms, such as lung carcinomas. It has been discovered that TS inhibitors increase cancer tissues' sensitivity to chemotherapy and radiation, increasing their vulnerability to these treatments. This article aims to provide a comprehensive insight into TS, examining its cellular details, detailing the heterocyclic moieties and molecular foundations, and providing a promising future outlook.

Folinic acid in colorectal cancer: esquire or fellow knight? Real-world results from a mono institutional, retrospective study

The stock of therapeutic weapons available in metastatic colorectal cancer (mCRC) has been progressively grown over the years, with improving both survival and patients' clinical outcome: notwithstanding advances in the knowledge of mCRC biology, as well as advances in treatment, fluoropyrimidine antimetabolite drugs have been for 30 years the mainstay of chemotherapy protocols for this malignancy. 5-Fluorouracil (5FU) seems to act differently depending on administration method: elastomer-mediated continuous infusion better inhibits Thymidylate Synthase (TS), an enzyme playing a pivotal role in DNA synthetic pathway. TS overexpression is an acknowledged poor prognosis predicting factor. The simultaneous combination of 5FU and folinate salt synergistically strengthens fluorouracil cytotoxic effect. In our experience, levofolinate and 5FU together in continuous infusion prolong progression free survival of patients suffering from mCRC, moreover decreasing death risk and showing a clear clinical benefit for patients, irrespective of RAS mutational status, primitive tumor side and metastases surgery.

Cyclic Peptides Acting as Allosteric Inhibitors of Human Thymidylate Synthase and Cancer Cell Growth.

Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness.

Establishment and Characterization of Pemetrexed-resistant NCI-H460/PMT Cells.

Pemetrexed (PMT) is a multitargeted antifolate agent that is used for treating patients with Non-Small Cell Lung Cancer (NSCLC). However, patients have presented clinical responses of drug resistance to PMT. This study aimed to explore the underlying mechanisms of PMT resistance in NSCLC cells. PMT-resistant NCI-H460/PMT cells were established by treating with PMT in a concentrationescalation manner. MTT assay and colony formation were performed to detect cell proliferation. Immunofluorescence was used to detect the expression of Ki-67. Transwell assay was performed to measure cell migration ability. qPCR and Western blot were used to detect the mRNA and protein expression levels of indicated genes. Small interfering RNAs (siRNA) were used to knockdown ATP binding cassette subfamily B member 1 (ABCB1) and Thymidylate Synthase (TYMS). This study showed that compared with the parental cells, the NCI-H460/PMT cells displayed weakened proliferation and enhanced cell mobility. In addition, the NCI-H460/PMT cells demonstrated cellular senescence, which might result in PMT resistance. The NCI-H460/PMT cells exhibited cross-resistance to other chemotherapeutics, including fluorouracil, paclitaxel, doxorubicin, etoposide and gemcitabine, possibly because of the upregulated expression of ABCB1. However, the ABCB1 knockdown by siRNA failed to eradicate PMT resistance. Moreover, TYMS, a target of PMT, was obviously upregulated in the resistant cells. The genetic silence of TYMS partially abrogated PMT resistance, suggesting that the overexpression of TYMS was a key resistant mechanism of PMT. The overexpression of TYMS was an important resistance mechanism of PMT for KRAS-mutated NCI-H460 cells. Cross-resistance to other chemotherapeutics should be considered in addressing PMT resistance.

Study of Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) Expressions on 5-Fluorouracil in Oral Squamous Cell Carcinoma

Background:The study aims to analyze Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) Expressions on 5-Fluorouracil in Oral Squamous Cell Carcinoma (OSCC). Methods:50 oral squamous cell carcinoma samples were taken from non-treated cancer patients at Hiroshima University Dental Hospital. The patients were investigated for TS, that included 36 males and 14 females. Additionally, 31 patients were evaluated for DPD that included 22 males and 9 females.Results:The samples had also undergone clinical and pathological evaluation, immunohistochemical staining, evaluation of immune-staining, enzymatic expression, and statistical analyses. Mean age of the population was 62.1 years. Conclusion: Over-expression of TS contributes significantly to the resistance of 5-FU treatment; while inhibition of intra-tumoral DPD increases the sensitivity level. TS levels are not only predictive of 5-FU response, but also prognostic in clinical value of non-treated cancer patients.

Association of thymidylate synthase (TYMS) polymorphism with adverse events (AEs) of 5-FU/capecitabine (CAP) in pts with GI cancers.

e14561Background: TYMS catalyzes methylation of dUMP to dTMP. As the sole de novo source of thymidylate in the cell it is an important target for 5-FU/CAP. Overexpression of TYMS has been linked to...

Thymidylate synthase: a predictive biomarker in resected colorectal liver metastases receiving 5-FU treatment.

To investigate the role of thymidylate synthase (TS) as a predictive biomarker in patients with resected colorectal liver metastases (CRLM). PubMed, EMBASE and Cochrane Library were queried up to June 2017. Meta-analysis was performed using random-effects model. Risk of bias was assessed using funnel plots. Six eligible studies were included, comprising a total of 542patients. Meta-analysis demonstrated a trend to reduced overall survival in patients with resected CRLM with TS overexpression, with a hazard ratio of 1.13 (95% CI: 0.99-1.29; p=0.08). In three studies where patients received systemic fluorouracil, the pooled hazard ratio was 2.25 (95% CI: 1.37-3.71; p=0.001). TS appears to be a clinically relevant predictive biomarker in patients with resected CRLM receiving systemic 5-FU.

Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging.

IntroductionInhibition of thymidylate synthase (TS) results in a transient compensatory “flare” in thymidine salvage pathway activity measureable with 18F-thymidine (FLT)- positron emission tomography (PET) at 2hrs. of therapy which may predict non-small cell lung cancer (NSCLC) sensitivity to TS inhibition.Materials and MethodsResistance to TS inhibition by pemetrexed was induced in NSCLC cell lines H460 and H1299 through TS overexpression. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed resistance confirmed with IC50 assays. The presence of a pemetrexed-induced thymidine salvage pathway “flare” was then measured using 3H-thymidine in both pemetrexed sensitive (H460 and H1299) and resistant (H460R, H1299R, CALU-6, H522, H650, H661, H820, H1838) lines in vitro, and validated with FLT-PET in vivo using H460 and H460R xenografts.ResultsOverexpression of TS induced pemetrexed resistance with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Thymidine salvage pathway 3H-thymidine “flare” was observed following pemetrexed in H460 and H1299 but not H460R, H1299R, CALU-6, H522, H650, H661, H820 or H1838 in vitro. Similarly, a FLT “flare” was observed in vivo following pemetrexed therapy in H460 but not H460R tumor-bearing xenografts.ConclusionsImaging of TS inhibition is predictive of NSCLC sensitivity to pemetrexed.

Abstract 1783: Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging

Abstract Background: Inhibition of thymidylate synthase (TS) results in a transient compensatory “flare” in thymidine salvage pathway activity which is measureable with 18F-thymidine (FLT) positron emission tomography (PET) at 2 hrs of exposure to therapy. Here we examine the predictive value of the FLT-PET measured “flare” for NSCLC sensitivity to pemetrexed, a commonly used TS inhibitor, in a preclinical model. Experimental Design: Resistance to pemetrexed therapy was induced in two sensitive human NSCLC cell lines, H460 and H1299, by overexpressing TS. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed-resistance confirmed with IC50 assays. The presence of a pemetrexed-induced DNA salvage pathway “flare” was then measured using 3H-thymidine in both pemetrexed-sensitive (H460 and H1299) and resistant (H460R and H1299R) lines in vitro, as well as inherently resistant NSCLC cell lines CALU6, H522, H650, H661, H820, H1838, and validated with FLT-PET in vivo using H460 and H460R xenografts. Results: Overexpression of TS induced resistance to pemetrexed in the H460 and H1299 with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Significant increases in DNA salvage pathway activity (“flare”) was observed at 2hrs of therapy in the pemetrexed-sensitive H460 and H1299 lines but not the resistant H460R and H1299R, CALU6, H522, H650, H661, H820, H1838 cell lines in vitro using 3H-thymidine assays. Similarly, FLT “flare” was observed in the pemetrexed-sensitive H460 xenograft tumors but not the pemetrexed-resistant H460R xenograft tumors with post-therapy increases in measured FLTmax of 58% (STD 12.1) and 10.8% (STD 7.3) respectively. Conclusions: FLT-PET imaging of TS inhibition may provide an early indicator of NSCLC sensitivity to pemetrexed. Citation Format: Xiao Chen, Yizeng Yang, Sharyn Katz. Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1783. doi:10.1158/1538-7445.AM2017-1783

Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) and Microsatellite Instability in Patients with Colorectal Cancer and Its Clinical Features.

Recently, a different type of microsatellite instability (MSI) instability designated 'elevated microsatellite alterations at selected tetranucleotide repeats' (EMAST) has been reported in several neoplasms, but its clinical implications remain unclear. We aimed to determine the relationships among EMAST, MSI and clinicopathologic characteristics, including oncologic outcomes, in colorectal cancer (CRC). We evaluated 100 sporadic CRC cases subjected to surgery using five markers (MYCL1, D9S242, D20S85, D8S321, and D20S82) for EMAST and the Bethesda panel for MSI status. Immunohistochemical detection of hMSH3, c-erbB2, EGFR and thymidylate synthase was performed. Clinical characteristics and prognostic relevance were assessed. We identified 22 EMAST-positive tumors (22.0%) and 32 MSI-high (MSI-H) tumors (32.0%). EMAST was more frequent in colon cancer than rectal cancer (p=0.033), and associated with MSI-H phenotype (p<0.001), low expression of hMSH3 (p=0.004), and overexpression of thymidylate synthase (p=0.006). Among the 38 MSI-L tumors, only one (4.5%) showed EMAST. Long-term oncologic results in terms of overall and disease-free survival were similar between EMAST and non-EMAST tumors. EMAST is more closely related to MSI-H than MSI-L or MSS status. The clinical and molecular characteristics of EMAST were distinct in terms of tumor location, thymidylate synthase expression, MSI status and hMSH3 expression. Our preliminary findings support the utility of EMAST as a new potential classifier in CRC.

Systemically Administered RNAi Molecule Sensitizes Malignant Pleural Mesotheliomal Cells to Pemetrexed Therapy.

Pemetrexed (PMX) is a key drug for the management of malignant pleural mesothelioma (MPM). However, its therapeutic efficacy is cruelly restricted in many clinical settings by the overexpression of thymidylate synthase (TS) gene. Recently, we emphasized the efficacy of locally administered shRNA designed against TS gene in enhancing the cytotoxic effect of PMX against orthotopically implanted MPM cells in tumor xenograft tumor model. Herein, we explored the efficiency of systemic, rather than local, delivery of TS RNAi molecule in sensitizing MPM cells to the cytotoxic effect of PMX. We here designed a PEG-coated TS shRNA-lipoplex (PEG-coated TS shRNA-lipoplex) for systemic injection. PEG modification efficiently delivered TS shRNA in the lipoplex to tumor tissue following intravenous administration as indicated by a significant suppression of TS expression level in tumor tissue. In addition, the combined treatment of PMX with systemic injection of PEG-coated TS shRNA-lipoplex exerted a potent antitumor activity in a s.c. xenograft tumor model, compared to a single treatment with either PMX or PEG-coated TS shRNA-lipoplex. Metastasis, or the spread, of mesothelioma substantially dedicates the effectiveness of treatment options. The systemic, in addition to local, delivery of tumor targeted anti-TS RNAi system we propose in this study might be an effective option to extend the clinical utility of PMX in treating malignant mesothelioma.

Autoantibodies against TYMS and PDLIM1 proteins detected as circulatory signatures in Indian breast cancer patients.

Breast cancer (BC) is the most common invasive cancer in women worldwide. Autoantibodies (AAbs) to tumor-associated antigens (TAAs) have a great potential for the development of diagnostic biomarkers in cancer. This study was performed to identify AAbs and cognate TAAs that may improve detection of this deadly disease. Serological proteome analysis of plasma samples of BC patients (N = 30) and healthy controls (N = 30) was performed to identify TAAs. Expressions of selected TAAs were also determined in breast tumor tissues (N = 10) by immunohistochemistry. An independent validation cohort (N = 124) was tested to determine diagnostic accuracy of selected AAbs titer by ELISA. Thymidylate synthase (TYMS) and C-terminal LIM domain protein 1 (PDLIM1) were found to react more specifically with plasma samples of BC patients. Both TAAs were also found to be significantly over expressed (p < 0.001) in breast tumor tissues compared to adjacent normal tissues. TYMS AAbs response was positively correlated (r = 0.778, p < 0.008) with TYMS overexpression in BC tissues. TYMS and PDLIM1 AAbs titers discriminated BC from controls with a sensitivity/specificity of 57.81%/95% and 73.44%/58.33%, respectively. High titers of both TYMS and PDLIM1 AAbs were significantly more prevalent in BC cases than controls. Our data recommends further investigations for evaluating their potential for BC detection.

Overexpression of thymidylate synthase (TYMS) is associated with aggressive tumor features and early PSA recurrence in prostate cancer.

Thymidylate synthase (TYMS) plays a role in DNA synthesis and is a target for 5-fluorouracil. In this study TYMS was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancers. TYMS expression was higher in neoplastic than in normal prostate epithelium and was detectable in 72.9% of 10,223 interpretable cancers. It was considered strong in 21.9%, moderate in 33.4% and weak in 17.6% of tumors. TYMS overexpression was associated with deletions at 5q21 (p < 0.0001), 6q15 (p < 0.0001) and 3p13 (p = 0.0083) and gradually increased with the total number of these deletions present in the respective cancer sample (p < 0.0001). TYMS expression was unrelated to PTEN deletions (p = 0.9535) but tightly linked to high Gleason grade, advanced pathological tumor stage and early PSA recurrence (p < 0.0001). The prognostic value of TYMS was independent from the ERG status and deletions at 3p13, 5q21, and 6q15. In multivariate analyses the prognostic role of TYMS expression was independent of Gleason grade, pT stage, preoperative PSA, pN stage, or resection margins. TYMS expression analysis might result in clinically useful information in prostate cancer. The striking link to some but not all chromosomal aberrations might suggest a mechanistical link with specific types of DNA damage.

Bioinformatic Analysis for the Validation of Novel Biomarkers for Cancer Diagnosis and Drug Sensitivity

Background: The genetic control of tumour progression presents the opportunity for bioinformatics and gene expression data to be used as a basis for tumour grading. The development of a genetic signature based on microarray data allows for the development of personalised chemotherapeutic regimes. Method: ONCOMINE was utilised to create a genetic signature for ovarian serous adenocarcinoma and to compare the expression of genes between normal ovarian and cancerous cells. Ingenuity Pathways Analysis was also utilised to develop molecular pathways and observe interactions with exogenous molecules. Results: The gene signature demonstrated 98.6% predictive capability for the differentiation between borderline ovarian serous neoplasm and ovarian serous adenocarcinoma. The data demonstrated that many genes were related to angiogenesis. Thymidylate synthase, GLUT-3 and HSP90AA1 were related to tanespimycin sensitivity (p=0.005). Conclusions: Genetic profiling with the gene signature demonstrated potential for clinical use. The use of tanespimycin alongside overexpression of thymidylate synthase, GLUT-3 and HSP90AA1 is a novel consideration for ovarian cancer treatment.

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

Prognostic significance of thymidylate synthase expression in pancreatic adenocarcinoma: A meta-analysis.

The prognostic significance of thymidylate synthase (TS) overexpression in pancreatic adenocarcinoma has been extensively investigated; however, data on the survival of patients with pancreatic adenocarcinoma remain equivocal. We performed a meta-analysis of previous studies to assess the effects of TS overexpression on the overall survival (OS) of patients with pancreatic adenocarcinoma, using hazard ratio (HR) with its 95% confidence interval (CI). A total of 5 studies, including 425 patients, were subjected to the final analysis. The pooled HR was 0.72 (95% CI: 0.41-1.25; Z=1.18, P=0.238), indicating that TS expression exerted no significant survival effect on patients with pancreatic adenocarcinoma. The combined HR was 0.46 (95% CI: 0.31-0.68; Z=3.95, P<0.001), limiting the analysis to the studies assessing R0 resection patients, which indicated that a high expression of TS was significantly correlated with better OS in patients with pancreatic adenocarcinoma who underwent R0 resection. This meta-analysis identified TS as an independent factor predicting favourable outcome following R0 curative resection in patients with pancreatic adenocarcinoma.

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