Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cells William Fazzone 1 , Peter M. Wilson 1 , Melissa J. LaBonte 1 , Heinz-Josef Lenz 2 and Robert D. Ladner 1 * 1 Department of Pathology, Norris Comprehensive Cancer Center/University of Southern California, Keck School of Medicine, Los Angeles, CA 2 Division of Medical Oncology, Sharon A. Carpenter Laboratory, Norris Comprehensive Cancer Center/University of Southern California, Keck School of Medicine, Los Angeles, CA Despite recent therapeutic advances, the response rates to chemo- therapy for patients with metastatic colon cancer remain at 50% with the fluoropyrimidine, 5-fluorouracil (5-FU), continuing to serve as the foundation chemotherapeutic agent for the treatment of this disease. Previous studies have demonstrated that overex- pression of thymidylate synthase (TS) is a key determinant of re- sistance to 5-FU-based chemotherapy. Therefore, there is a signifi- cant need to develop alternative therapeutic strategies to over- come TS-mediated resistance. In this study, we demonstrate that the histone deacetylase inhibitors (HDACi) vorinostat and LBH589 significantly downregulate TS gene expression in a panel of colon cancer cell lines. Downregulation of TS was independent of p53, p21 and HDAC2 expression and was achievable in vivo as demonstrated by mouse xenograft models. We provide evidence that HDACi treatment leads to a potent transcriptional repression of the TS gene. Combination of the fluoropyrimidines 5-FU or FUdR with both vorinostat and LBH589 enhanced cell cycle arrest and growth inhibition. Importantly, the downstream effects of TS inhibition were significantly enhanced by this combination includ- ing the inhibition of acute TS induction and the enhanced accumu- lation of the cytotoxic nucleotide intermediate dUTP. These data demonstrate that HDACi repress TS expression at the level of transcription and provides the first evidence suggesting a direct mechanistic link between TS downregulation and the synergistic interaction observed between HDACi and 5-FU. This study pro- vides rationale for the continued clinical evaluation of HDACi in combination with 5-FU-based therapies as a strategy to overcome TS-mediated resistance. ' 2009 UICC Key words: colon cancer; thymidylate synthase; histone deacetylase inhibitors; fluoropyrimidines; 5-fluorouracil; vorinostat; LBH589 For over 50 years, the fluoropyrimidine class of anticancer agents such as 5-fluorouracil (5-FU) have served as the foundation for the treatment of colon cancer. Chemotherapy-based approaches for the treatment of colon cancer have improved significantly in recent years with 5-FU used in combination with newer agents such as oxaliplatin, irinotecan, cetuximab and bevacizumab. These combinations now demonstrate response rates up to 50%. 1–3 However, the clinical efficacy of these treatments is hindered due to intrinsic or acquired drug resistance, and novel strategies to overcome resistance are of great clinical importance. Fluoropyrimidines such as 5-FU and FUdR induce cellular toxicity by inhibiting the enzyme thymidylate synthase (TS) and by incorporating fluoronucleotides into RNA and DNA. The TS enzyme converts dUMP to TMP and serves as the sole de novo source of thymidylate and is essential for DNA synthesis. 5-FU inhibits TS by the formation of a stable ternary complex con- sisting of the 5-FU metabolite fluorodeoxyuridine monophos- phate (FdUMP), the folate cofactor 5,10-methylene tetrahydrofo- late, and the TS enzyme leading to thymidylate depletion, cell cycle arrest and apoptosis. 1 FUdR is metabolized primarily to FdUMP and has been reported to induce more TS-specific effects when compared to 5-FU. Several clinical studies, con- ducted by multiple independent laboratories, have demonstrated that elevated TS gene expression is associated with resistance to 5-FU-based therapy in part due to TS overexpression. 4–6 Several factors have been shown to contribute to alterations in TS gene expression leading to 5-FU resistance including various poly- morphic variations within the TS gene that contribute to differ- ential expression in addition to elevated expression of key regu- latory transcription factors such as E2F-1. 7–10 Additional studies have shown that TS protein is acutely induced following 5-FU administration due to increased protein stability and translational derepression. 11,12 Despite advances in our understanding of the molecular factors that contribute to the cytotoxicity of 5-FU, 50% of patients with advanced colorectal cancer do not respond to current 5-FU-based therapy. Therefore, novel strat- egies to improve response rates and overcome 5-FU resistance are of great clinical importance. Histone deacetylase inhibitors (HDACi) have recently emerged as potent and selective anticancer agents. These agents demon- strate pleiotropic anticancer activities by the inhibition of histone deacetylases (HDACs), leading to changes in the acetylation status of both histone and nonhistone proteins including tubulin, Hsp90 as well as multiple transcription factors that can alter protein func- tion. 13–15 HDAC inhibition results in modulation of 2–10% of genes, promotion of differentiation, inhibition of cell cycle pro- gression, induction of apoptosis and suppression of tumor angio- genesis. 16 DNA microarray profiling was used to study the effects of multiple HDACi on gene expression within bladder and breast cancer cell lines. This study identified TS as one of the most heav- ily downregulated genes following treatment with HDACi. 17 Recent reports have extended this initial observation and demon- strated that the HDACi PXD101 has synergistic effects on inhibi- tion of cell growth when combined with 5-FU in colon and gastric cancer cells. 18,19 These authors postulated that HDACi-induced acetylation of Hsp90 destabilized TS protein and that TS mRNA may be downregulated through a transcriptional mechanism. 18 Based on these initial observations, we sought to elucidate the mechanistic basis for downregulation of TS mRNA by HDACi within colon cancer cell lines and examine the combinatorial effects of clinically relevant HDACi with fluoropyrimidines on co- lon cancer cell lines to provide further rationale for the combina- tion of these agents in the clinic. In this study, we demonstrate that 2 clinically relevant agents of the hydroxamate class of HDACi, vorinostat (suberoylanilide hydroxamic acid, SAHA) and LBH589, cause a potent downregu- lation of TS gene expression within multiple colon cancer cell lines. Downregulation of TS protein was further demonstrated The first two authors contributed equally to this work. *Correspondence to: Department of Pathology, Norris Comprehensive Cancer Center/University of Southern California, Keck School of Medi- cine, 1441 Eastlake Avenue, Room 5322, Los Angeles, CA 90033, USA. Fax: 11-323-8653099. E-mail: rladner@usc.edu Additional Supporting Information may be found in the online version of this article. Grant sponsor: V-Foundation–AACR Grant for Translational Research, Littlefield–ACCR Grant for Metastatic Colon Cancer Research. Received 3 September 2008; Accepted after revision 17 February 2009 DOI 10.1002/ijc.24403 Published online 27 February 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 463–473 (2009) ' 2009 UICC Publication of the International Union Against Cancer