Expression and pharmacological inhibition of thymidylate synthase and Src kinase in nonsmall cell lung cancer Paolo Ceppi, Ida Rapa, Marco Lo Iacono, Luisella Righi, Jessica Giorcelli, Marisa Pautasso, Andrea Bille `, Francesco Ardissone, Mauro Papotti and Giorgio V. Scagliotti Department of Clinical and Biological Sciences, University of Turin at San Luigi Hospital, Orbassano, Italy The combination of cytotoxic chemotherapy with signaling pathway inhibitors represents a potential strategy to improve the treatment of nonsmall cell lung cancer (NSCLC). Thymidylate synthase (TS) is an enzyme essential for DNA synthesis, and its overexpression has been associated with the reduced sensitivity to antifolate agents. Src is a tyrosine kinase that modulates the cytotoxicity of cancer cells after drug treatment, and in vitro data indicate that its inhibition could revert the resistance to TS-inhibiting drugs. Our study investigated the significance of TS and Src expression in NSCLC tissues, and the effects of their pharmacological inhibition in cell lines. In tumor and normal tissues from 94 resected NSCLC patients, TS and Src transcript levels were found positively correlated (R S 5 0.66), associated with patients smoking history and overall survival. At multivariate analysis, TS gene expression was an independent prognostic factor (relative risk (RR) 5 1.78, from 1.16 to 2.72; p < 0.01). Immunohistochemical detection in tumor specimens confirmed that Src kinase activation, evaluated by phospho- specific antibody, was associated to a higher TS expression. In cell lines, dasatinib, a Src-inhibiting agent, synergistically enhanced pemetrexed-cytotoxicity of A549 cells, as evaluated by MTT and apoptosis assays. The biological explanation for this interaction was based on the upregulation of TS messenger RNA and protein levels induced by pemetrexed, which was significantly prevented by dasatinib cotreatment. The data of our study suggest that TS and Src may belong to a common pathway that bears prognostic significance in NSCLC, and that Src represents a potential target to improve the efficacy of TS-inhibiting agents. Cytotoxic chemotherapy is the standard of care for locally advanced and metastatic nonsmall cell lung cancer (NSCLC) even though the efficacy of the currently available therapeutic armamentarium is limited, 1,2 and new therapeutic strategies are clearly needed to improve patients quality of life, response rates and overall survival. Thymidylate synthase (TS) is the enzyme which catalyzes the reaction that provides the sole de novo intracellular source of deoxythymidine monophosphate, or thymidylate, which is essential for DNA replication and repair. 3,4 TS pro- tein and messenger RNA (mRNA) levels have been found elevated in many human cancers, and correlated with poor prognosis in patients with colorectal, 5 breast, 6 head and neck, 7 pancreatic 8 and in NSCL cancers. 9,10 Transfection of catalytically active TS has been shown to induce a trans- formed phenotype in human cells, as manifested by foci for- mation, anchorage independent growth and tumor formation in nude mice, suggesting that TS itself could be considered as an oncogene. 11 TS is also the cellular target of 5-fluorouracil (5-FU) 4 and of pemetrexed. 12 Pemetrexed is a multitargeted antifolate that prevents the formation of DNA and RNA, blocking the growth of cancer cells, by inhibiting the activity of four enzymes in the folate pathway, being TS the most im- portant target. 13–15 In lung cancer, it was previously reported an higher TS mRNA and protein level in squamous cell car- cinoma, as opposed to adenocarcinoma, 10 and a 10-fold higher TS expression in small-cell lung cancer (SCLC) as compared to NSCLC 16 and to carcinoid tumors. 17 More recently, it was shown a consistency between the aforemen- tioned findings and TS enzymatic activity in lung cancer cell lines of different histotypes, 18 and these findings led to a ra- tionale interpretation of the outcome of the clinical trials which investigated the role of pemetrexed in NSCLC and SCLC. 19–21 As molecular strategies aimed at TS inhibition, it has been shown to improve the in vitro effects of peme- trexed; 13 in our study, we investigated one of the molecular mechanisms that could regulate TS expression in lung cancer cells. Src is a tyrosine kinase (TK) involved in many aspects Key words: nonsmall cell lung cancer, thymidylate synthase, Src tyrosine kinase, pemetrexed Additional Supporting Information may be found in the online version of this article. Potential conflicts of interest: M.P. and G.V.S. received honoraria from Eli Lilly. Grant sponsor: Regione Piemonte, Ricerca Sanitaria Finalizzata; Grant number: 14631/DB20.01 DOI: 10.1002/ijc.26188 History: Received 19 Jan 2011; Accepted 28 Apr 2011; Online 26 May 2011 Correspondence to: Paolo Ceppi, Department of Clinical and Biological Sciences, University of Turin. Regione Gonzole 10, 10043 Orbassano, ITALY, Tel.: þ39-011-9026719, Fax: þ39-011-9026753, E-mail: paolo.ceppi@unito.it; p-ceppi@northwestern.edu Cancer Cell Biology Int. J. Cancer: 130, 1777–1786 (2012) V C 2011 UICC International Journal of Cancer IJC