British Journal of Cancer (2010) 102, 553 – 560 & 2010 Cancer Research UK All rights reserved 0007 – 0920/10 $32.00 www.bjcancer.com The role of folate receptor alpha (FRa) in the response of malignant pleural mesothelioma to pemetrexed-containing chemotherapy *,1 1,3 1 2 1 1,3 1,3 1 JE Nutt , ARA Razak , K O’Toole , F Black , AE Quinn , AH Calvert , ER Plummer and J Lunec 1 2 Northern Institute for Cancer Research, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK; Department of Pathology, Royal 3 Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK; Northern Centre for Cancer Care, Freeman Hospital, Freeman Road, Newcastle upon Tyne NE7 7DN, UK BACKGROUND: The standard treatment of choice for malignant pleural mesothelioma is chemotherapy with pemetrexed and platinum, but the clinical outcome is poor. This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FRa). METHODS: Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50% (GI50). FRa expression was determined by western blotting and that of FRa, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT–PCR. Immunohistochemistry for FRa was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed. RESULTS: A wide range of GI50 values was obtained for the cell lines, H2452 cells being the most sensitive (GI50 22 nM) and RS5 cells having a GI50 value greater than 10 mM.NoFRa protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRa was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRa and the outcome of pemetrexed treatment, and no significant difference between histological subtypes. CONCLUSION: Response to treatment with pemetrexed does not depend on the presence of FRa. Translational Therapeutics British Journal of Cancer (2010) 102, 553–560. doi:10.1038/sj.bjc.6605501 www.bjcancer.com Published online 5 January 2010 & 2010 Cancer Research UK Keywords: Folate receptor a; mesothelioma; pemetrexed; cell lines; immunohistochemistry Malignant pleural mesothelioma (MPM) is an aggressive tumour of survival and quality of life, compared with single agent cisplatin, the pleura and usually has a poor prognosis. It is increasing in and suggested that this be used as front-line chemotherapy in many countries worldwide as a result of widespread exposure to MPM patients (Vogelzang et al, 2003). Response rate in this study asbestos in the past and is expected to peak around 2015 in the was 41%, with a median survival benefit of 2.8 months, but relapse United Kingdom (Hodgson et al, 2005). The disease affects more rates remain high and long-term survival is poor; therefore, men than women in a ratio of 5 : 1 and tends to have a long latency improved patient selection for treatment in this disease is still period of 20–40 years after asbestos exposure, affecting people in required. the fifth to seventh decade of life. Approximately 80% of MPM is Pemetrexed is a multitargeted antifolate that inhibits several attributed to exposure to asbestos fibres, although some tumours enzymes important in folate metabolism, the main action being on are ‘spontaneous’ with no evidence of asbestos exposure. The thymidylate synthetase (TS). Inhibition of TS leads to depleted outcome of patients with MPM is generally poor, with a median levels of thymidine, which are crucial to DNA synthesis. survival of 6–12 months. Most patients are unsuitable for radical Pemetrexed also inhibits, to a lesser degree, glycinamide surgery. Radiation therapy has been shown to alleviate pain in the ribonucleotide formyltransferase (GARFT) (Shih et al, 1997). Once majority of treated patients, but the duration of symptom control in the cell, pemetrexed undergoes polyglutamation, often resulting is short lived, hence chemotherapy is generally the treatment of in a tri- or penta-glutamate tail being added to the parent drug. choice. Clinical trials have shown a partial response rate of 32% This process is facilitated by the enzyme folyl-poly glutamate using a combination of pemetrexed and carboplatin (Hughes synthetase (FPGS), for which pemetrexed has a high affinity, and et al, 2002). A phase III trial using pemetrexed with cisplatin results in cellular drug retention. The affinity of pemetrexed to TS significantly improved response rates, time to progression, overall is also increased by at least 60 times in the polyglutamated form, compared with the parent drug (Hanauske et al, 2001). Folic acid and antifolates enter cells by three mechanisms. The *Correspondence: Dr JE Nutt; E-mail: [email protected] reduced folate carrier (RFC) is a bidirectional anion exchanger that Revised 23 November 2009; accepted 26 November 2009; published has a high affinity for reduced folate co-factors and antifolates, but online 5 January 2010 a low affinity for folic acid. It has a neutral pH optimum and is FRa and pemetrexed in mesothelioma JE Nutt et al 554 ubiquitously expressed (Whetstine et al, 2002). The folate receptor 50% growth inhibition (GI50) for each cell line was calculated using alpha (FRa) is an energy-dependent high-affinity, low-capacity GraphPad Prism software. folate-binding protein that delivers folates and antifolates into Cell lysates required for western blotting were prepared after cells. The low capacity is due to endocytosis of the receptor that washing cells with ice-cold PBS and addition of lysis buffer has to return to the cell surface in order to function. Expression is (62.5 mM Tris/HCl pH 6.8, 2% sodium dodecyl sulphate and 10% limited to specific tissues and is expressed in a characteristic glycerol). distribution, for example, on syncytiotrophoblastic cells of the placenta and the proximal convoluted tubule of the kidney Western blotting (Weitman et al, 1992; Mantovani et al, 1994). However, FRa has been reported to be highly expressed in some malignancies: in 90% Western blotting was carried out to determine the presence and of non-mucinous epithelial ovarian cancers (Toffoli et al, 1997) level of expression of FRa protein. Cell lysates were prepared and and in 72% of malignant mesotheliomas (Bueno et al, 2001). Folate 20 mg aliquots of protein (calculated from quantification using the receptor alpha has been believed to be important for the Pierce BCA protein assay) from each sample were loaded on a 4– physiological transport of folic acid, but not generally for 20% gradient polyacrylamide gel (Invitrogen). After SDS–PAGE antifolates (Kamen and Smith, 2004). However, there is evidence and electroblotting (Towbin et al, 1979), membranes were treated to suggest that FRa may have an important role in folate and for 1 h with TBS-Tween buffer containing 5% milk to block non- antifolate transport under certain circumstances. Studies per- specific binding and then incubated with a previously described formed on vulval epithelial cell lines A431 and A431-FBP, a highly and validated monoclonal mouse FRa primary antibody (Smith expressing FRa transfectant, showed that A431-FBP was approxi- et al, 2007) for 1 h at room temperature. Secondary HRP-conjugated mately three-fold more sensitive to pemetrexed than the isogenic antibody (goat anti-mouse, Dako, Ely, UK) was used to detect Translational Therapeutics non-FRa-expressing cell line when cells were maintained in a the primary antibodies and antibody-labelled protein bands were physiological level of folate (20 nM leucovorin) and was 14-fold visualised by enhanced chemiluminescent detection (ECL) (GE more sensitive when maintained in sub-physiological levels (1 nM Healthcare, Little Chalfont, UK). Equal loading of protein was leucovorin) (Theti and Jackman, 2004). Membrane transport of verified using a mouse anti-tubulin antibody (Sigma–Aldrich). folates has also been recently reported to occur through a IGROV1 (an ovarian cancer cell line) cell lysate was used as a ubiquitously expressed proton-coupled folate transporter (PCFT) positive control and Jurkat cell lysate was used as a negative (Wang et al, 2003; Zhao et al, 2008), a low-pH transporter with a control for FRa. high affinity for pemetrexed. The contribution of FRa to pemetrexed transport is likely to be Quantitative RT–PCR significant in cells in which FRa is highly expressed. In these situations, transport of pemetrexed via FRa may enhance delivery RNA was extracted from cells using the Qiagen RNAeasy minikit of the drug to the tumour and potentially enhance response. (Qiagen, Hilden, Germany) and reverse transcribed using TaqMan Recently, a new monoclonal FRa antibody has been developed that reverse transcriptase reagents (Applied Biosystems, Warrington, UK). can be used for immunohistochemistry on formalin-fixed paraffin- A volume of 2.5 ml cDNA was used for quantitative real-time PCR embedded (FFPE) tissues (Smith et al, 2007) and therefore be more in a total volume of 10 ml, repeated in triplicate, using the TaqMan widely used on stored and more readily transportable tumour gene expression assay Hs00357143_g1 for FRa, Hs00953344_m1 specimens. This study investigates the growth inhibitory effects of for RFC, Hs00611082_m1 for PCFT and Hs99999901_s1 for 18S pemetrexed in eight human mesothelioma cell lines and their FRa (all from Applied Biosystems), as well as universal mastermix. status. It also reports the response to treatment with pemetrexed in The expected amplicon sizes were 89, 106, 72 and 187, respectively. mesothelioma patients in relation to FRa immunohistochemistry PCR was performed as follows: 501C for 2 min, 951C for 10 min, of the tumours. and 40 cycles of 951C for 15 s and 601C for 1 min using the ABI Prism 7900HT sequence detection system (Applied Biosystems).