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Novel Opportunities for Thymidylate Metabolism As a Therapeutic Target

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Novel Opportunities for Thymidylate Metabolism As a Therapeutic Target 3029 Novel opportunities for thymidylate metabolism as a therapeutic target Peter M. Wilson,1 William Fazzone,1 small-molecule inhibitor to dUTPase represents a viable Melissa J. LaBonte,1 Jinxia Deng,2 strategy to improve the clinical efficacy of these mainstay Nouri Neamati,2 and Robert D. Ladner1 chemotherapeutic agents. [Mol Cancer Ther 2008; 7(9):3029–37] 1Department of Pathology, Norris Comprehensive Cancer Center, Keck School of Medicine, and 2Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California Introduction The fluoropyrimidine 5-fluorouracil (5-FU) is widely used in the treatment of a range of cancers, including breast Abstract cancers, and cancers of the aerodigestive and gastrointes- For over 40 years, the fluoropyrimidine 5-fluorouracil tinal tract (1). However, 5-FU has had the greatest effect (5-FU) has remained the central agent in therapeutic and is arguably the most successful drug approved to date regimens employed in the treatment of colorectal cancer for the treatment of colorectal cancer. Throughout 50 years and is frequently combined with the DNA-damaging of clinical development, the response rate of advanced agents oxaliplatin and irinotecan, increasing response colorectal cancer chemotherapy using 5-FU and 5-FU-based rates and improving overall survival. However, many combinations has improved from 10% to 15% to 40% to patients will derive little or no benefit from treatment, 50% primarily due to the introduction of efficacious combi- highlighting the need to identify novel therapeutic targets nation partners such as the topoisomerase I inhibitor to improve the efficacy of current 5-FU-based chemother- irinotecan and the platinum agent oxaliplatin and deter- apeutic strategies. dUTP nucleotidohydrolase (dUTPase) mination of optimal drug scheduling and administration catalyzes the hydrolysis of dUTP to dUMP and PPi, pro- (2–4). Novel biological agents, such as the monoclonal viding substrate for thymidylate synthase (TS) and DNA antibodies cetuximab, which targets the epidermal growth synthesis and repair. Although dUTP is a normal interme- factor receptor and bevacizumab an inhibitor of vascular diate in DNA synthesis, its accumulation and misincor- endothelial growth factor, have recently shown additional poration into DNA as uracil is lethal. Importantly, uracil clinical benefit when included in 5-FU-based regimens in misincorporation represents an important mechanism of metastatic colorectal cancer through suppression of recep- cytotoxicity induced by the TS-targeted class of chemo- tor-mediated tumor processes (5, 6). However, despite therapeutic agents including 5-FU. A growing body of these improvements, approximately one-half of patients evidence suggests that dUTPase is an important mediator treated with 5-FU-based therapies will derive no benefit, of response to TS-targeted agents. In this article, we pre- highlighting the need for the identification of novel sent further evidence showing that elevated expression of therapeutic targets and strategies to overcome the frequent dUTPase can protect breast cancer cells from the expan- occurrence of drug resistance. sion of the intracellular uracil pool, translating to reduced 5-FU continues to remain the mainstay of therapeutic growth inhibition following treatment with 5-FU. We regimens employed in the treatment of colorectal cancer therefore report the implementation of in silico drug and other gastrointestinal malignancies. However, 5-FU development techniques to identify and develop small- continues to prove itself an efficacious agent in additional molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU cancers including breast cancer. Two pivotal clinical trials prodrug capecitabine remain central agents in the treat- have recently reported capecitabine to be an efficacious ment of a variety of malignancies, the clinical utility of a combination partner for novel targeted agents approved for breast cancer (7, 8). In this article, we show that induced dUTP nucleotidohydrolase (dUTPase) expression in a Received 3/26/08; revised 6/16/08; accepted 6/19/08. tetracycline (Tet)-repressible MCF-7 breast cancer cell line Grant support: NIH grant R21 5R21CA104796-3. Wright Foundation, suppresses dUTP pool expansion and increases resistance Whittier Foundation, and Concern Foundation. to 5-FU. Importantly, we also show that dUTPase expres- The costs of publication of this article were defrayed in part by the sion in breast cancer specimens shows marked variation payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to similar to our previous observations in colon cancer. In indicate this fact. summary, dUTPase represents an unexploited therapeutic Requests for reprints: Robert D. Ladner, Department of Pathology, Norris target and the identification of effective inhibitors has the Comprehensive Cancer Center, Keck School of Medicine, University of potential to improve the efficacy of 5-FU-based chemo- Southern California, Room 5322, 1441 Eastlake Avenue, Los Angeles, CA 90089. Phone: 323-865-3116; Fax: 323-865-0522. therapies in a wide variety of cancers. To this end, we have E-mail: [email protected] generated a dUTPase pharmacophore model using in silico Copyright C 2008 American Association for Cancer Research. drug development techniques as a means to identify novel doi:10.1158/1535-7163.MCT-08-0280 small-molecule antagonists to dUTPase. Mol Cancer Ther 2008;7(9). September 2008 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. 3030 dUTPase: A Novel Therapeutic Target Materials and Methods loading. Horseradish peroxidase signal was detected using Compounds and Reagents HyGlo and Hyblot film (Denville Scientific) and developed 5-FU, fluorodeoxyuridine (FUdR), and paclitaxel were on a Hope Micromax film processor (Hope X-Ray). purchased from Sigma. Growth Inhibition Assay Cell Culture MCF-7 pTet-off cells were transfected in the presence or A The human breast adenocarcinoma MCF-7 pTet-off cell absence of 0.5 g/mL doxycycline and growth inhibition line was obtained from BD Clontech and grown in was measured as previously described (9) using CellTiter DMEM supplemented with 10% Tet-approved fetal 96 AQueous One Solution (Promega). Cells were exposed bovine serum (BD Clontech) with penicillin/streptomycin to increasing concentrations of 5-FU for 72 h. Absorbance and sodium pyruvate (Invitrogen). Cells were maintained was measured using a SpectraMax 190 microplate reader in a humidified Forma incubator (Thermoscientific) at (Molecular Devices) at 490 nm, with drug-treated cells j compared with untreated controls set at 100%. Statistical 37 C with 5% CO2. Overexpression of dUTPase significance was determined using a two-tailed unpaired t MCF-7 pTet-off cells were seeded on 6-cm plates, and Student’s test (GraphPad). Immunohistochemistry 3 h after plating, the cells were washed with PBS and fresh Immunohistochemistry using the DUT415 monoclonal growth medium was added. After 24 h, cells were trans- A fected with 2 Ag pTre-Tight:DUT-N for 6 h and washed in antibody (2 g/mL) was conducted on formalin-fixed, PBS and the appropriate medium was added; to suppress paraffin-embedded breast adenocarcinoma tissue samples the inducible expression of dUTPase, doxycycline was using methods as described previously (12). added to growth medium containing Tet-approved fetal bovine serum at a final concentration of 0.5 Ag/mL. Results and Discussion Twenty-four hours post-transfection, cells were plated for 5-FU Mechanism of Action the appropriate assay and allowed to adhere for 24 h before Following entry into the cell, 5-FU is converted to its medium containing 5-FU, FUdR, or paclitaxel was added. active metabolite, fluoro-dUMP whose primary mechanism Overexpression of dUTPase was confirmed using both of action is inhibition of thymidylate synthase (TS) by Western blotting and enzyme activity assay. formation of a ternary complex with the methyl cofactor dUTPase ActivityAssay 5,10-methylene tetrahydrofolate. This blocks the de novo Cells were harvested and protein was isolated and synthesis of thymidylate resulting in perturbations in A quantified as per Western blotting. Total protein (25 g) nucleotide pools and severe disruption of DNA synthesis A was normalized to a 20 L reaction volume with PBS/ and repair and ultimately leads to lethal DNA damage (1). protease inhibitor. Relative dUTPase activity was deter- Additional mechanisms of action include the incorporation mined as described previously (9) and is expressed as fold of toxic fluoronucleotides into both DNA and RNA and the change compared with an identical transfection in the expansion of the intracellular dUTP pool and subsequent A presence of 0.5 g/mL doxycycline. misincorporation into DNA (1). The 5-FU prodrug capeci- dUTP Accumulation Assay tabine has the convenience of oral administration and has MCF-7 pTet-off cells were treated with specified concen- shown equivalent efficacy as both a single agent and in trations of 5-FU, FUdR, and paclitaxel for indicated times combination with oxaliplatin for the first-line treatment of  6 and harvested, and 3 10 cells were analyzed for metastatic colorectal cancer (13, 14). Capecitabine is nucleotide pool content using the assay developed by absorbed intact through the gastrointestinal mucosa where Sherman and Fyfe (10) modified to detect levels
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