Downregulation Of Thymidylate Synthase Research Articles

Capsaicin Acts Through Reducing P38 MAPK-Dependent Thymidylate Synthase Expression to Enhance 5-Fluorouracil-Induced Cytotoxicity in Human Lung Cancer Cells

Capsaicin, an ingredient of green and red bell peppers, shows anticancer activity in several malignant cell lines. Thymidylate synthase (TS) is a well-validated anticancer drug target in non-small cell lung cancer (NSCLC) cells. However, whether capsaicin and 5-fluorouracil (5-FU) induce synergistic cytotoxicity in NSCLC cells by regulating TS expression is unclear. This study investigated the cytotoxicity of capsaicin and 5-FU co-treatment on two hoursuman lung adenocarcinoma cell lines, H520 and H1703, and the underlying mechanisms. Capsaicin decreased TS expression in a p38 mitogen-activated protein kinase (MAPK) inactivation–dependent manner in H520 and H1703 cells. Enhancement of p38 MAPK activity by transfection with constitutive active mitogen-activated protein kinase kinase six vectors increased TS expression and cell survival. In addition, capsaicin and 5-FU co-treatment enhanced synergistic cytotoxicity and inhibited cell growth associated with TS downregulation and p38 MAPK inactivation in H520 and H1703 cells. Capsaicin and 5-FU co-treatment did not affect the cellular content of capsaicin. These results show that capsaicin may be combined with 5-FU to treat NSCLC.

Tacalcitol increases the sensitivity of colorectal cancer cells to 5-fluorouracil by downregulating the thymidylate synthase

5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells.We showed that PRI-2191 induces the CDKN1A (gene encoding p21Waf1/Cip1) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action.In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog.

High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase.

The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53-null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild-type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.

Abstract 3141: Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase

Abstract Although non-small cell lung cancer (NSCLC) cells with anaplastic lymphoma kinase (ALK)-rearranged initially show a dramatic response to crizotinib, these cells eventually develop resistance to crizotinib. This resistance is caused by secondary mutations and copy number gain in the ALK gene. However, other resistance mechanisms through activation of the bypass tracts have yet to be clearly elucidated. To investigate the mechanisms of acquired resistance to ALK fusion-directed treatment in NSCLC, we generated crizotinib-resistant NSCLC cell lines in vitro through chronic exposure of an ALK fusion NSCLC line (SNU2292) to crizotinib. Interestingly, the resultant SNU2292-CR cells maintained activation of epidermal growth factor receptor (EGFR) and expressed increased levels of transforming growth factor alpha (TGF-α), an EGFR ligand. Additionally, we found that thymidylate synthase (TS) was overexpressed in SNU2292-CR cells compared with the parental cells. We showed reduction of TS enhanced synergistic inhibition of cell proliferation when cetuximab, an EGFR inhibitor, was combined with pemetrexed, a TS inhibitor. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against crizotinib-resistant cells by downregulating TS expression. Taken together, these results support a potential role of activation of the bypass tracts in acquired resistance to ALK-directed treatment in ALK-rearranged NSCLC, and provide insights into strategies for preventing and/or overcoming this resistance in patients Citation Format: Hwang-Phill Kim, Sae-Won Han, Sang-Hyun Song, Tae-You Kim. Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase [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 3141. doi:10.1158/1538-7445.AM2017-3141

Safety and antitumor activity of arsenic trioxide plus infusional 5-fluorouracil, leucovorin, and irinotecan as second-line chemotherapy for refractory metastatic colorectal cancer: A pilot study from South India.

After failing oxaliplatin-based first-line chemotherapy (CT), approximately 4%-21% of patients with metastatic colorectal cancer (mCRC) respond to irinotecan-based second-line treatment. Earlier studies have demonstrated that arsenic trioxide (ATO) can significantly resensitize resistant colon cancer to 5-fluorouracil (5-FU) by downregulating thymidylate synthase (TS). We hypothesized that a combination of ATO with infusional 5-FU, leucovorin, and irinotecan (FOLFIRI) regimen in mCRC patients refractory to first-line FOLFOX/CAPOX could further improve the outcome of second-line CT. Patients were administered ATO 0.15 mg/kg/day on days 1-2 along with FOLFIRI regimen at standard doses every 2 weeks, until disease progression, unacceptable toxicity, or patients' refusal. Responses to CT were reported according to RECIST 1.1. Adverse events were classified based on CTCAE version 4.0. Between September 2016 and July 2017, 17 patients with refractory mCRC were treated with this investigational combination. The median age was 49 years; 13 males and 4 females; ECOG PS 0-1/2, 14/3. The most common site of metastases was liver (n = 11) followed by peritoneum (n = 7) and number of involved metastatic sites 1-2/≥3, 9/8. After 6 cycles of CT, overall response rate and disease control rate were 17.6% and 82.4%, respectively (complete remission = 0, partial remission = 3 patients, stable disease = 11 patients). Median progression-free survival was 5.3 months (95% confidence interval [CI]: 4.3-7.0) and median overall survival was 9 months (95% CI: 7.4-10.5) from the initiation of ATO plus FOLFIRI. The toxicities were as follows: Grade 1/2 toxicity: fatigue (7 patients), constipation (2), and nausea and vomiting (2); Grade 3 toxicity: fatigue (3), neutropenia (2), febrile neutropenia (1), diarrhea (2), and QTc prolongation (1). No patient experienced Grade 4 toxicities. The addition of ATO to FOLFIRI regimen as second-line CT in patients with refractory mCRC offered an encouraging antitumor effect at the cost of manageable toxicity.

Abstract 4659: Folic acid-metabolizing enzymes regulate 5-fluorouracil anticancer activity on colorectal cancer cell lines

Abstract Background: In colorectal cancer chemotherapy, the current standard of care includes 5-fluorouracil (5-FU) and leucovorin (LV) combination therapy. To understand the role of folic acid-metabolizing enzymes in the LV-mediated enhancement of 5-FU's antitumor activity, we suppressed the expression of genes related to folic acid metabolism and evaluated the resultant effect on the proliferation inhibitory effect of 5-fluoro-2′-deoxyuridine (FdUrd). Method: We downregulated the expression of thymidylate synthase (TS), folate receptor 1 (FOLR1), methylenetetrahydrofolate reductase (MTHFR), methylenetetrahydrofolate dehydrogenase (MTHFD), dihydrofolate reductase (DHFR), and phosphoribosylglycinamide formyltransferase (GART) by lipofection of small interfering RNA (siRNA) in DLD-1 and HCT116 cells, and calculated the relative change in IC50 values on 48 hr exposure to FdUrd in comparison with the corresponding IC50 values in control siRNA-treated cells. In cells with TS downregulation, LV-mediated enhancement of sensitivity to FdUrd was also evaluated. Sensitivity to FdUrd was determined using the crystal violet staining method. Results: Treatment with siRNA downregulated the expression of folic acid-metabolizing enzymes. LV enhanced the sensitivity to FdUrd in control siRNA-treated cells in a dose-dependent manner. However, in cells with TS downregulation, LV did not enhance the sensitivity to FdUrd. Downregulation of folic acid-metabolizing enzymes, except TS, decreased the efficacy of FdUrd, although there was little decrease in FdUrd efficacy owing to MTHFR downregulation. Conclusion: The level of TS expression determined the LV requirement, and downregulation of folic acid-metabolizing enzymes, which impaired the folic acid cycle, decreased the efficacy of 5-FU. Comparison of the IC50 values in target siRNA-treated cells and control siRNA-treated cells.DLD-1 cellsHCT116 cellsexpression (%)*IC50 (μM)fold changeexpression (%)*IC50 (μM)fold changeNon-silencing Control siRNA100.000.671.00100.000.251.00TS10.580.010.0114.840.030.12FOLR19.148.6512.9125.122.5910.36MTHFR25.441.432.1348.750.883.52MTHFD9.662.734.0715.061.997.96DHFR8.524.146.1817.231.706.80GART18.905.388.037.102.469.84Values are means (n = 3). *: Expression (%) is the expression at the beginning of FdUrd treatment (at 48 hr after siRNA treatment). Citation Format: Hiroshi Tsukihara, Kenta Tsunekuni, Teiji Takechi. Folic acid-metabolizing enzymes regulate 5-fluorouracil anticancer activity on colorectal cancer cell lines. [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 4659.

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

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

Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model.

Malignant pleural mesothelioma (MPM) is an incurable cancer with an increasing incidence. Currently, pemetrexed (PMX)-based chemotherapy is the mainstay of chemotherapy for MPM, however, the outcome of PMX-based chemotherapy in patients with MPM is dismal. RNA interference (RNAi) technology has been considered as an effective tool to substantially enhance the therapeutic efficacy of chemotherapeutic agents in many preclinical and clinical settings. In this study, therefore, we investigated whether non-viral anti-thymidylate synthase RNAi embedded liposome (TS shRNA lipoplex) would effectively guide the downregulation of TS in human malignant mesothelioma MSTO-211H cells. Consequently, it enhanced the antitumor effect of PMX both in vitro and in vivo. TS shRNA effectively enhanced the in vitro cell growth inhibition upon treatment with PMX via downregulating TS expression in the MSTO-211H cell line. In in vivo orthotopic tumor model, the combined treatment of PMX and TS shRNA lipoplex efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged mouse survival, compared to each single treatment. Our findings emphasize the pivotal relevance of RNAi as an effective tool for increasing the therapeutic efficacy of PMX, a cornerstone in the treatment regimens of MPM, and thereby, raising the possibility for the development of a novel therapeutic strategy, combination therapy of TS-shRNA and PMX, that can surpass many of the currently applied, but less effective, therapeutic regimens against lethal MPM.

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

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

Gossypol sensitizes the antitumor activity of 5-FU through down-regulation of thymidylate synthase in human colon carcinoma cells.

5-Fluorouracil (5-FU) is the basic chemotherapeutic agent used to treat colon cancer. However, the sensitivity of colon cancer cells to 5-FU is limited. Gossypol is a polyphenolic extract of cottonseeds. The purpose of this study was to investigate the activities and related mechanism of gossypol alone or in combination with 5-FU against human colon carcinoma cells. The IC50 of gossypol or/and 5-FU in vitro was tested by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the drug interaction was analyzed using the CalcuSyn method. Cell apoptosis was determined using presidium iodide staining and flow cytometric analysis. Western blotting was used to determine the expression of proteins. Transient transfection method was used to silence protein. The IC₅₀ at 48 h of gossypol in colon cancer cells was 26.11 ± 1.04 μmol/L in HT-29 cells, 14.11 ± 1.08 μmol/L in HCT116 cells, and 21.83 ± 1.05 μmol/L in RKO cells. When gossypol was combined with 5-FU, a synergistic cytotoxic effect was observed in HT-29 cells, HCT116 cells, and RKO cells compared with treatment with gossypol or 5-FU alone. The Western blotting results indicated that gossypol down-regulated thymidylate synthase (TS) rather than thymidine phosphorylase protein expression. Furthermore, the mTOR/p70S6K1 signaling pathway was inhibited in gossypol-treated colon cancer cells, and consequently, cyclin D1 expression was decreased, suggesting an additional mechanism of the observed antiproliferative synergistic interactions. All the observation was confirmed by silencing TS and inactivating the mTOR/p70S6K1 signaling pathway by rapamycin, both of which increased the chemo-sensitizing efficacy of 5-FU. These findings suggest that gossypol-mediated down-regulation of TS, cyclin D1, and the mTOR/p70S6K1 signaling pathways enhances the anti-tumor effect of 5-FU. Ultimately, our data exposed a new action for gossypol as an enhancer of 5-FU-induced cell growth suppression.

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.

MiR-203 enhances chemosensitivity to 5-fluorouracil by targeting thymidylate synthase in colorectal cancer.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that play important roles in diverse biological processes, including chemoresistance. However, the molecular mechanism as to how miR-203 modulates the chemosensitivity to 5-fluorouracil (5-FU) in colorectal cancer is poorly known. In the present study, we found that miR-203 was downregulated in the 5-FU-resistant cell line LoVo/5-Fu, and was inversely correlated with the extent of 5-FU chemoresistance. Cytotoxicity assay showed that the inhibition of miR-203 expression enhanced 5-FU chemoresistance in colorectal cancer cells, while miR-203 overexpression increased 5-FU chemosensitivity. We then validated that thymidylate synthase (TYMS) was a direct target of miR-203 and miR-203 suppressed TYMS protein levels. Silencing of TYMS enhanced 5-FU chemosensitivity, similar to the roles of miR-203. Finally, we discovered that miR-203 increased the inhibitory effects of 5-FU on tumor growth in vivo. Overall, our data indicate that miR-203 enhances 5-FU chemosensitivity via the downregulation of TYMS in colorectal cancer and provide important insight into the mechanism of 5-FU resistance in colorectal cancer patients. More important, the present study suggests that miR-203 has the potential as a therapeutic strategy for 5-FU-resistant colorectal cancer.

Downregulation of thymidylate synthase and E2F1 by arsenic trioxide in mesothelioma.

Malignant pleural mesothelioma is a global health issue. Arsenic trioxide (ATO) has been shown to suppress thymidylate synthase (TYMS) in lung adenocarcinoma and colorectal cancer, and induce apoptosis in acute promyelocytic leukemia. With TYMS as a putative therapeutic target, the effect of ATO in mesothelioma was therefore studied. A panel of 5 mesothelioma cell lines was used to study the effect of ATO on cell viability, protein expression, mRNA expression and TYMS activity by MTT assay, western blot, qPCR and tritium-release assay, respectively. The knockdown of TYMS and E2F1 was performed with a specific siRNA. Phosphatidylserine externalization and mitochondrial membrane depolarization were measured by AnnexinV and JC-1 staining respectively. The invivo effect of ATO was studied using a nude mouse xenograft model. Application of ATO demonstrated anticancer effects in the cell line model with clinically achievable concentrations. Downregulation of TYMS protein (except H226 cells and 1.25µM ATO in H2052 cells) and mRNA expression (H28 cells), pRB1 (H28 cells) and E2F1 and TYMS activity (except H226 cells) were also evident. E2F1 knockdown decreased cell viability more significantly than TYMS knockdown. In general, thymidine kinase 1, ribonucleotide reductase M1, c-myc and skp2 were downregulated by ATO. p-c-Jun was downregulated in H28 cells while upregulated in 211H cells. Phosphatidylserine externalization, mitochondrial membrane depolarization, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bak and cleaved caspase-3 were observed. In the H226 xenograft model, the relative tumor growth was aborted, and E2F1 was downregulated while cleaved caspase-3 was elevated and localized to the nucleus in the ATO treatment group. ATO has potent antiproliferative and cytotoxic effects in mesothelioma invitro and invivo, partially mediated through E2F1 targeting (less effect through TYMS targeting). There is sound scientific evidence to support the clinical application of ATO in treatment of mesothelioma.

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

Abstract Purpose Objectives: Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Ganetespib is a potent functional inhibitor of HSP90 currently under evaluation in clinical trials. We tested the hypothesis that HSP90 functional inhibition by ganetespib can inhibit cell cycle progression and downregulate TS levels in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50 nM for 24 hours. Western blot and Real time-PCR analyses were carried out to determine the effect of ganetespib on different signal molecules involved in cell cycle arrest and DNA synthesis. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of ganetespib. Pre and post ganetespib treatment tumor biopsies from patients with rectal cancer on a phase I trial of ganetespib were analyzed using RT-PCR. Results: Treatment with ganetespib inhibited cyclin D1, pRb and E2F-1 at the transcriptional and translational level and induced p21, leading to G0/G1 cell cycle arrest. HSP90 knockdown inhibited E2F-1 and TS protein and mRNA levels in both cell lines. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. Similar effects were observed in tumors from animals treated with ganetespib. Further, ganetespib treatment significantly inhibited the tumor growth. Inhibition of cyclin D1, Rb, E2F-1, TS and upregulation of p21 at transcriptional levels were observed in post treatment rectal tumor biopsies obtained from patients receiving ganetespib. Conclusion: In this study, we present preclinical and clinical data to support the use of HSP90 inhibitors in colorectal cancer based on their ability to reduce expression of proteins important for DNA repair and cell cycle progression leading to cell cycle arrest and tumor shrinkage. Accordingly, combining HSP90 inhibitors with chemotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Ganji Purnachandra Nagaraju, Field F. Willingham, Kevin E. Woods, Patrick Sullivan, Jerome C. Landry, Roberto Diaz, Bassel F. El-Rayes. Functional inhibition of HSP90 induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1308. doi:10.1158/1538-7445.AM2014-1308

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

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

Downregulation of thymidylate synthase with arsenic trioxide in lung adenocarcinoma

Thymidylate synthase (TYMS) is an important chemotherapeutic target in non-small cell lung cancer (NSCLC). Arsenic trioxide (ATO) has been shown to suppress TYMS in a colonic cancer model. We examined the effects of TYMS suppression by ATO in lung adenocarcinoma. A panel of 4 lung adenocarcinoma cell lines was used to determine the effects of ATO treatment on cell viability, TYMS expression (protein and mRNA), E2F1 protein expression and TYMS activity. TYMS knockdown and overexpression were performed. Tumor growth inhibition invivo was studied using a nude mouse xenograft model. ATO showed antiproliferative effects with clinically achievable concentrations (around 1.1-6.9µM) in 4 lung adenocarcinoma cell lines. Downregulation of TYMS protein and mRNA expression, reduced TYMS activity, and suppressed E2F1 expression were demonstrated in lung adenocarcinoma with ATO. Cell viability was reduced by 15-50% with TYMS knockdown. Overexpression of TYMS led to a 2.7-fold increase in IC50 value with ATO treatment in H358 cells, but not in H23 cells. Using a xenograft model with H358 cell line, relative tumor volume was reduced to 44% that of the control following 8days of treatment with 7.5mg/kg ATO, and associated with significant downregulation of TYMS protein expression. In conclusion, ATO has potent invitro and invivo activity in lung adenocarcinoma, and is partially mediated by transcriptional downregulation of TYMS.

Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.

Simvastatin enhances the chemotherapeutic efficacy of S-1 against bile duct cancer

Simvastatin has inhibitory effects on cancers. The present study aimed to investigate the interactive effects between simvastatin and S-1 against bile duct cancer and its mechanisms. The effects of simvastatin and 5-fluorouracil (5-FU) alone or in combination on the growth and apoptosis of the human cholangiocarcinoma cell line EGI-1 and the gallbladder carcinoma cell line Mz-ChA-1 cells were evaluated in vitro. Real-time PCR and western blot were used to determine E2F-1 and thymidylate synthase (TS) expressions in the treated cells. Tumoricidal efficacy of simvastatin and S-1 was further investigated in a subcutaneous bile duct cancer model in NOD/SCID mice. Simvastatin enhanced the cytotoxicity of 5-FU on bile duct cancer cells in vitro. IC50 of 5-FU alone was 4.34 μmol/l for EGI-1 and 13.9 μmol/l for MZ-ChA-1, whereas it decreased markedly to 0.90 and 2.95 μmol/l, respectively, when combined with simvastatin. The Chou and Talalay combination index of 5-FU and simvastatin was 0.41 and 0.40 at IC50 for EGI-1 and MZ-ChA-1, respectively. Simvastatin alone or plus 5-FU significantly suppressed E2F-1 and TS expressions in EGI-1 and MZ-ChA-1. Simvastatin plus 5-FU induced greater proportion of apoptotic cells on both EGI-1 and MZ-ChA-1, with an increase in cleaved caspase-3 levels, compared with simvastatin or 5-FU alone (all P < 0.05). Simvastatin plus S-1 induced greater tumor inhibition than simvastatin or S-1 alone with E2F-1/TS downregulation in vivo (all P < 0.05). Simvastatin and S-1 exerted synergistic effects against bile duct cancer, which might be mediated by E2F-1/TS downregulation. The combination could be a reasonable regimen in the management of bile duct cancer.

Phase I study of the HDAC inhibitor vorinostat in combination with capecitabine in a biweekly schedule in advanced breast cancer.

154 Background: Synergy between histone deacetylase inhibitors and 5-fluorouracil is thought to be due to down regulation of thymidylate synthase. Study objectives are to assess maximum tolerated dose (MTD), dose limiting toxicities (DLT) and objective response rate of vorinostat (V) in combination with capecitabine (C). Secondary aims are pharmacodynamics of V. Methods: Cohorts of 3-6 patients (pts) were enrolled on a 3+3 dose-escalation phase to assess MTD of escalating doses of oral C (1500/1800/2000mg) with V 200 mg BID on days 1-7 and 15-21 of a 28 d cycle. 10 pts enrolled on dose expansion phase and treated at MTD. Pts received a “run in” treatment of V for 5 d and pre/post V biopsy (bx) collected when feasible. DLT was defined as ≥ grade 3 non hematological toxicity, grade 4 thrombocytopenia, grade 4 neutropenia &gt; 5 d, grade 4 febrile neutropenia requiring hospitalization, QTc &gt;500ms,or treatment delay &gt; 2 weeks from toxicity. Microarray analysis was performed using Illumina HT-12 gene arrays on pre/post bx from 4 pts. Results: 23 ptswith median age 51 (range 33-69) were treated: 8 pts at 1500/200mg, 5 pts at 1800/200mg, and 10 pts treated on dose expansion phase at 1500/200mg. Median # of prior lines of chemotherapy was 2 (range 0-7). 3 pts are still on study. Median # of cycles on study were 2 (range 1-42). Cycle 1 DLT was grade 3 fatigue in 2 pts treated on 1800/200mg, and in 1 pt treated on 1500/200mg. Other grade 3/4 toxicities are summarized in the Table. 14 pts are evaluable for response (12 pts at 1500/200mg and 2 pts at 1800/200mg). No objective responses were seen, 3 pts had stable disease &gt; 6 months (1 pt completed 42 cycles). All post V bx converged on principal components analysis. Changes in pathways involved in extracellular matrix and TGFb pathway were noted. Conclusions: DLT of C concomitant with V was fatigue. MTD was 1500 mg C combined with 200 mg V BID. Combination has modest clinical activity. Gene expression profiles suggest consistent pathway modulation with V. Clinical trial information: NCT00719875. [Table: see text]

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.