Thymidylate Synthase mRNA Expression Research Articles

Genomic and immunological features of microsatellite instability in colon cancer.

Microsatellite instability (MSI) is closely related to the prognosis and therapy response of colon cancer. Colon cancer patients with MSI show resistance to 5-Fluorouracil (5-FU) but sensitivity to immunosuppressive checkpoint inhibitors (ICIs). The relevant mechanism behind the opposite response remains unclear. Multi-omics research data of colon cancer patients were acquired from The Cancer Genome Atlas (TCGA) database, GEO database, and DAFI dataset. Transcriptome data were normalized to gene expression data through the R software package "Limma". Somatic mutations data were analyzed and visualized through the R software package "maftools". CIBERSORT algorithm was used to estimate the relative proportion of 22 infiltrating immune cell types. We demonstrated MSI patients showed both overexpressed immune checkpoints (mRNA level) and activated tumor-infiltrating lymphocytes (TILs), which may explain the satisfying response of ICIs. The additionally, we also demonstrated MSI promoted the mRNA expression of thymidylate synthase (TYMS) through regulating its copy number variation. As a main target of 5-FU, overexpressed TYMS promoted the resistance of 5-FU. Furthermore, we demonstrated MSI patients showed significantly increased somatic mutations compared with microsatellite stability (MSS) patients, except APC, TP53, and KRAS mutations. The substitutions and location of somatic mutations in different genes were at variance between MSS and MSI patients. In conclusion, our research determined mechanisms of MSI associated treatment response, and may provide potential value for improving the survival of colon cancer patients.

Plasma thymidylate synthase and dihydrofolate reductase mRNA levels as potential predictive biomarkers of pemetrexed sensitivity in patients with advanced non-small cell lung cancer.

BackgroundHigh levels of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expression in tumour tissues are an indicator of ineffective responses to pemetrexed-based chemotherapy in various tumours, including non-small cell lung cancer (NSCLC). However, tumour tissues are highly heterogeneous, so a single biopsy may not reflect genetic alterations during disease progression. This study investigated the potential use of plasma TS and DHFR mRNA levels as biomarkers for predicting sensitivity to pemetrexed-based chemotherapy.MethodsPlasma samples were obtained from 245 patients with advanced NSCLC and 30 healthy donors. Total RNA was extracted from the plasma samples, and TS and DHFR mRNA levels were determined via real-time PCR. TS and DHFR mRNA levels between cancer patients and healthy controls were compared. The association between plasma TS and DHFR mRNA levels and tumour response to pemetrexed/cisplatin chemotherapy was analysed.ResultsThe plasma TS and DHFR mRNA levels decreased in patients with advanced NSCLC compared with healthy controls. Moreover, plasma TS and DHFR mRNA levels negatively correlated with tumour response to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. Overall survival time was prolonged in patients with low TS mRNA expression compared with those with high TS mRNA expression, although the difference was not statistically significant.ConclusionsLow expression levels of plasma TS and DHFR mRNA confer increased tumour sensitivity to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. The results suggested that plasma TS and DHFR mRNA levels are promising biomarkers for choosing patients who are likely to respond and benefit the most from pemetrexed-based chemotherapy.

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

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

Abstract 5189: Evaluation of thymidylate synthase mRNA expression in circulating tumor cells from non-metastatic colon and rectal patients

Abstract Background: colorectal cancer is the third leading cause of cancer death in the United States. With different behaviors, rectal cancer represents worse prognosis, even in the locally advanced setting, with therapy following three steps, consisting of neoadjuvant chemoradiation, surgery and adjuvant chemotherapy. For non-metastatic colon cancer, the main treatment is surgery and then, patients can or not undergo adjuvant chemotherapy. Studies have shown that circulating tumor cells (CTCs) may be involved in metastatization process. Thymidylate synthase (TYMS) acts in the 5-Fluorouracil metabolism, and when overexpressed, can confer resistance to treatment, which is commonly used for neoadjuvant treatment (rectal cancer) and in combination with oxaliplatin for adjuvant treatment (colon cancer). Excision repair cross complementation Group 1 (ERCC1) is a protein involved in damage DNA repair process that is described to confer resistance to platinum-based chemotherapy. Objective: evaluate the TYMS messenger RNA (mRNA) expression in CTCs isolated from patients with locally advanced colon and rectal cancers; and its predictable value in treatment resistance. For patients with colon cancer, we also evaluated the ERCC1 protein expression in order to verify the adjuvant therapy resistance related to oxaliplatin. Methods: We collected 10 mL of blood before the beginning of treatment and processed it on ISET® (Rarecells) device. RNAscope® Technology is a novel chromogenic in situ hybridization (CISH) assay for detection of target RNA within intact cells. The expression of TYMS target gene was performed using the kit from ACDbio, as described on the protocol, but standardized to cytology. Results: there were included 12 patients of each type of tumor. We analyzed samples from 12 rectal cancer patients, and found 10 with overexpression of TYMS mRNA in CTCs. Curiously, of the two patients who had TYMSneg mRNA expression; one had complete pathological response and the other substantial down-staging. In the positive cases, 50% presented response (partial or complete) and 50% present no response and systemic progression. This result will better understood when we perform the evaluation of the protein TYMS. Among the samples from colon cancer patients, we found 8 out 12 as CTCs TYMSpos mRNA expression. Unfortunately, our recent data does not allow us to do analysis of recurrence. However, one of the positive cases had recurrence. Among the 4 TYMSneg, one had recurrence. This can be explained by the positive immunocytochemical expression of the ERCC1 protein, which confers resistance to oxaliplatin. Our next steps are to expand the CISH experiments in a larger group of patients as well as to evaluate the TYMS protein expression in the same cases. Conclusion: our preliminary results point molecular analysis of CTCs as predictor marker of colon/rectal cancer treatments. Citation Format: Bianca Troncarelli Flores, Emne Ali Abdallah, Virgílio Souza e Silva, Celso Abdon Mello, Samuel Aguiar, Renata Mayumi Takahashi, Vanessa Silva Alves, Bruna Elisa Kupper, Ludmilla T. Chinen. Evaluation of thymidylate synthase mRNA expression in circulating tumor cells from non-metastatic colon and rectal patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5189.

Antitumor effect of lapatinib and cytotoxic agents by suppression of E2F1 in HER2‑positive breast cancer.

The human epidermal growth factor receptor2 (HER2)‑targeting agent, lapatinib, combined with oral fluoropyrimidine capecitabine, has been previously demonstrated to be an effective treatment option for patients with trastuzumab‑resistant HER2‑positive metastatic breast cancer. To investigate the molecular mechanisms associated with the interactions between lapatinib and capecitabine, the effect of treatment with lapatinib and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K) inhibitors on the expression of E2F transcription factor 1 (E2F1) and thymidylate synthase (TS), which is associated with an increased response to 5‑fluorouracil (5‑FU)‑based chemotherapy, was determined in HER2‑positive breast cancer cells. The results of reverse transcription‑quantitative polymerase chain reaction demonstrated that administration of lapatinib and PI3K inhibitors decreased the mRNA expression of TS and E2F1, a transcription factor that promotes TS gene expression, in SKBR3 and T47D cell lines. Furthermore, treatment with lapatinib and PI3K inhibitors also suppressed the mRNA expression of ribonucleotide reductase M1 subunit (RRM1), an important determinant of gemcitabine resistance, and DNA topoisomerase II‑α (TOP2A), a molecular target of anthracyclines, in SKBR3 and T47D cell lines. Western blot analysis further demonstrated that the phosphorylation of Akt was inhibited by lapatinib, and the results of the MTT assay revealed that the combination of lapatinib with either 5‑FU or gemcitabine demonstrated synergistic antitumor effects, whereas a combinatory treatment of lapatinib with epirubicin, a typical anthracycline drug, exhibited antagonistic antitumor effects in HER2‑positive breast cancer cells. These results indicate that the synergistic antitumor effects exhibited by combinatory treatment of lapatinib with capecitabine may be induced via the suppression of E2F1‑mediated TS expression.

P1.02-044 Relationship between RET Rearrangement and Thymidylate Synthase mRNA Expression in Non-Small Cell Lung Cancer Tissues

P1.02-044 Relationship between RET Rearrangement and Thymidylate Synthase mRNA Expression in Non-Small Cell Lung Cancer Tissues

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

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

Prevalence of thymidylate synthase gene 5'-untranslated region variants in an Argentinean sample.

Thymidylate synthase (TYMS) is a key enzyme in nucleotide synthesis and therefore, an important target of many chemotherapeutic agents. Expression of TYMS mRNA is thought to be modulated by a 28-bp tandem repeat polymorphism within its 5'-untranslated region, raising the question of this variant's utility in predicting the efficacy and toxicity of cancer treatment regimens. The aim of the present research was to describe the distribution of this TYMS polymorphism in the Argentinean population. A total of 199 randomly selected DNA samples from healthy volunteers were analyzed using polymerase chain reaction and polyacrylamide gel electrophoresis. The 2R and 3R alleles were present in 47.74 and 52.26% of samples, respectively, with frequencies of 21.6 (43), 52.3 (104), and 26.1% (52) recorded for the 2R/2R, 2R/3R, and 3R/3R genotypes, respectively. No significant difference regarding gender was observed. Our prevalence data are similar to those reported for other Caucasian populations. This opens a discussion concerning the reference population valid for comparisons and the clinical importance of this genotyping test as an additional tool in personalized medicine.

P2.03a-007 Pem/CBP/Bev Followed by Pem/Bev in Hispanic Patients with NSCLC: Outcomes According to Combined Score of TS, ERCC1 and VEGF Expression

To evaluate the efficacy and safety of pemetrexed, carboplatin and bevacizumab (PCB) followed by maintenance pemetrexed and bevacizumab (PB) in chemotherapy-naïve patients with stage IV non-squamous non-small cell lung cancer (NSCLC) through the influence of thymidylate synthase (TS), ERCC1 and VEGF mRNA expression on several outcomes. The primary endpoints were the overall response rate (ORR), progression-free survival (PFS) and overall survival (OS). Patients were administered pemetrexed (500 mg/m2), carboplatin (AUC, 5.0 mg/ml/min) and bevacizumab (7.5 mg/kg) intravenously every three weeks for up to four cycles. Maintenance pemetrexed and bevacizumab was administered until disease progression or unacceptable toxicity. One hundred forty-four Hispanic patients with a median follow-up of 13.8 months and a median number of maintenance cycles of 6 (range, 1- 32) were assessed. The ORR among the patients was 66% (95% CI, 47% to 79%). The median progression-free and overall survival (OS) rates were 7.9 months (95% CI, 5.9-10.0 months) and 21.4 months (95% CI, 18.3 to 24.4 months), respectively. Median TS, ERCC1 and VEGF mRNA levels were 1.45 (range, 0.17–2.52), 0.58 (range, 0.44-1.20), and 2.72 (range, 1.84-3.21), respectively. OS was significantly higher in patients with the lowest TS mRNA levels [29.6 months (95% CI 26.2-32.9) compared with those with higher levels 9.3 months (95% CI 6.6-12.0); p=0.0001]. ERCC1 mRNA levels also influenced the OS [median for ERCC1 mRNA˂0.58 28.7 months (95% CI 26.3-31.2) vs. ERCC1 mRNA˃0.58 11.1 months (95% CI 9.6-12.7); p=0.0001] as well as VEGF mRNA levels [median OS for VEGF mRNA˂2.72 26.4 months (95% CI 22.8-30.0) vs. VEGF mRNA˃2.72 18.2 months (95% CI 8.4-27.9); p=0.009]. TS mRNA did not influence treatment response, however the ORR was significantly higher in patients with low levels of ERCC1 (p = 0.003) and elevated VEGF (p = 0.005). Multivariate analysis found that TS mRNA levels (p=0.0001), VEGF mRNA levels (p=0.007) and PS (p=0.014) were independent prognostic factors. Overall, PCB followed by maintenance pemetrexed and bevacizumab was in Hispanic patients with non-squamous NSCLC. This regimen was associated with prolonged OS, particularly in patients with low TS, ERCC1 and VEGF mRNA expression. These biomarkers alone or in combination may be useful to assess the prognosis of patients with NSCLC treated with CBP/Pem/Bev.

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

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

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues.

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed.

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.

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 LB-220: Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS)

Abstract Background: Tumor resistance to chemotherapy is a major challenge in cancer therapeutics today. 5-Fluorouracil (5-FU) is one of the most commonly used cancer therapeutic agents. The Forkhead box transcription factor M1 (FOXM1) has been shown to play vital roles in cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal carcinoma and has been implicated in drug resistance in breast cancer. Thymidylate synthase (TS) enzyme is an important precursor in DNA synthesis. The mechanism of action of 5-FU has been associated with TS. High TS expression levels in tumors are generally related to 5-FU resistance. In this study we investigated the role of FOXM1 in 5-FU resistance and the therapeutic potential of the FOXM1 inhibitor thiostrepton (a thiazole antibiotic) in combination with 5-FU in colorectal cancer. We further analyzed the relation between TS and FOXM1 gene expressions in colorectal cancer. Methods and Results: HCT116, DLD1, and HT29 cells were treated with 5-FU to detect the protein, mRNA expression of FOXM1 and TS. This resulted in a decrease in TS and FOXM1 expression in p53 wild type HCT116 cells but not in p53 mutant DLD1 or HT29. We also observed that FOXM1 expression was significantly correlated with TS expression by thiostrepton treatment. The IC50 of 5-FU was determined by SRB assay and was found to be 0.92ug/mL, 0.45ug/mL and 2.8ug/mL, while thiostrepton 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 effect in all 3 colorectal cancer cells. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cells to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). Three 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 decrease 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 (TK1). 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 E2F1, E2F2 and E2F3 was also shown in both cell lines. Conclusion: FOXM1 plays a critical role in 5-FU drug resistance by regulating 5-FU target genes such as TS, TK1. Inhibition of FOXM1 acts synergistically with 5-FU in colorectal cancer and warrants further investigation as a new treatment strategy. Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Francesco A. Mauri, Eric W. Lam, Laura M. Kenny. Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS). [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 LB-220. doi:10.1158/1538-7445.AM2014-LB-220

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

Multiplex real-time PCR for RRM1, XRCC1, TUBB3 and TS mRNA for prediction of response of non-small cell lung cancer to chemoradiotherapy.

This study was aimed to establish a novel method to simultaneously detect expression of four genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1 (XRCC1), thymidylate synthase (TS) and class III β-tubulin (TUBB3), and to assess their application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to chemoradiotherapy. We have designed four gene molecular beacon (MB) probes for multiplex quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS mRNA expression in paraffin-embedded specimens from 50 patients with advanced or metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin- based first-line treatment were analyzed. These molecular beacon probes could specially bind to their target genes in homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have apparently higher response rates to chemoradiotherapy compared with those with high levels of RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly associated with chemotherapy response (p>0.05). A method of simultaneously detecting four molecular markers was successfully established and applied for evaluation of chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.

Final Results of a Phase II Study of S-1 in Patients with Cytokine-refractory Metastatic Renal Cell Carcinoma

A planned primary analysis of a Phase II study of S-1 demonstrated that the drug was active and tolerable in Japanese patients with cytokine-refractory metastatic renal cell carcinoma. Furthermore, pharmacogenomic analysis suggested that low expression of thymidylate synthase mRNA may have been associated with clinical outcome in terms of overall response rate and progression-free survival. Here, we report the results of the final analysis assessing the efficacy and safety of S-1 including overall survival. Patients with renal cell carcinoma were eligible if they had had at least one regimen of cytokine for metastatic disease. S-1 was orally administered on Days 1-28 of a 42-day cycle until disease progression. The primary endpoint was the overall response rate, and the secondary endpoint included progression-free survival, overall survival and safety. A total of 45 patients were treated with S-1 and were fully assessable for efficacy and safety. At the final analysis, a response was seen in 11 patients (overall response rate, 24.4%; 95% confidence interval: 12.9-39.5%), including two patients who achieved a complete response. The final median progression-free and overall survival were 9.2 and 42.8 months, respectively. The safety profile of S-1 was favorable. It was suggested that there was no relation between overall survival and the expression level of thymidylate synthase. This final analysis confirms that S-1 treatment is effective and safe in patients with cytokine-refractory renal cell carcinoma.

Cinnamaldehyde/chemotherapeutic agents interaction and drug-metabolizing genes in colorectal cancer

Cinnamaldehyde is an active monomer isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which is known to possess marked antitumor effects in vitro and in vivo. The aim of the present study was to examine the potential advantages of using cinnamaldehyde in combination with chemotherapeutic agents commonly used in colorectal carcinoma (CRC) therapy, as well as to investigate the effect of cinnamaldehyde on chemotherapeutic-associated gene expression. The synergistic interaction of cinnamaldehyde and chemotherapeutic agents on human CRC HT-29 and LoVo cells was evaluated using the combination index (CI) method. The double staining with Annexin V conjugated to fluorescein-isothiocyanate and phosphatidylserine was employed for apoptosis detection. The expression of drug-metabolizing genes, including excision repair cross‑complementing 1 (ERCC1), orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), breast cancer susceptibility gene 1 (BRCA1) and topoisomerase 1 (TOPO1), all in HT-29 and LoVo cells, with or without the addition of cinnamaldehyde, was examined by quantitative polymerase chain reaction (PCR). Cinnamaldehyde had a synergistic effect on the chemotherapeutic agents cytotoxicity in HT-29 and LoVo cells. In addition, cinnamaldehyde suppressed BRCA1, TOPO1, ERCC1 and TS mRNA expression, except for OPRT expression, which was markedly upregulated. Our findings indicate that cinnamaldehyde appears to be a promising candidate as an adjuvant in combination therapy with 5-fluorouracil (5-FU) and oxaliplatin (OXA), two chemotherapeutic agents used in CRC treatment. The possible mechanisms of its action may involve the regulation of drug‑metabolizing genes.

Effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells

The study investigated the effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells. Six human colorectal cancer cells were exposed to pemetrexed, gefitinib, and their combination. Antitumor effects were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Thymidylate synthase (TS) mRNA expression and EGFR mutation were studied by real-time RT-PCR and DNA sequence determination. Pharmacological interaction was studied using the combination index method. Cell cycle distribution and apoptosis were determined by flow cytometry. Activity assay was performed to assess the effects of drugs on TS activity, and Western blot was performed to assess the protein expression of pEGFR, pAKT, and pERK 1/2. Six colorectal cancer cells are all sensitive to pemetrexed, and TS gene expression of cells was negatively related to pemetrexed sensitivity. The cytotoxic synergism was observed in concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib. The combination of pemetrexed and gefitinib modulated cell cycle and induced apoptosis. Pemetrexed combined with gefitinib decreased TS mRNA expression and in situ activity. Pemetrexed induced an EGFR-mediated activation of the phosphatidylinositol 3-kinase/AKT and ERK pathway, which was inhibited by gefitinib. Pemetrexed is a promising agent, and pemetrexed combined with gefitinib has a significantly synergistic effect on colorectal cancer cells, which seems to present a strategy of pemetrexed combined with EGFR-TKIs in colorectal cancer treatment.

Substitution of anti-androgens and tegafur-uracil combination therapy for castration-resistant prostate cancer: Results of a multi-center randomized phase II study

We conducted this study to determine whether substitution with anti-androgen (SOA) and tegafur-uracil (a pro‑drug of 5-FU) combination therapy is more effective than SOA alone after relapse from initial hormonal therapy. Patients who were histologically confirmed and relapsed after initial hormonal therapy were included. All patients were randomly allocated into two groups: SOA alone (group A) or SOA combined with tegafur-uracil (group B). The mRNA expression of four enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phospho-ribosyltransferase (OPRT) and thymidine phosphorylase (TP), in prostate cancer cells was analyzed by quantitative reverse-transcription polymerase chain reaction. Fifty-two patients were enrolled in this study. The median age was 77 (range: 47-92) years. The PSA response rate in group B (61.5%) tended to be higher compared to that in group A (34.6%) (p=0.095). Group B (median: 15.9 months) had a significantly longer time to PSA progression (TTP) compared to group A (6.4 months) (p=0.014). In patients with a lower TS expression or a higher OPRT expression, group B demonstrated a higher PSA response rate compared to group A (p=0.019 and p=0.041, respectively). In addition, in the patients with a lower TS expression, group B demonstrated a significantly longer TTP compared to group A (p=0.018). There were no severe adverse events in either treatment group. After relapse from initial hormonal therapy, SOA combined with tegafur-uracil is effective and well tolerated. The TS mRNA expression level may be a predictive factor for this combination therapy.