Cytochrome P 450 Profile of Colorectal Cancer : Identification ofMarkers of Prognosis

Purpose: The cytochromes P450 (P450) are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, carcinogens, and endogenous compounds.The purpose of this study was to define the P450 profile of colorectal cancer and establish the prognostic significance of expression of individual P450s in colorectal cancer. Experimental Design: Immunohistochemistry for apanelof 23P450swas doneona colorectal cancer tissuemicroarray consisting of 264 primary colorectal cancers, 91lymphnodemetastasis, and 10 normal colorectal samples. The intensity of immunoreactivity in each sample was established by light microscopy. Results: The most frequently expressed form of P450 in normal colonwas CYP3A4. In primary colorectal cancer, several P450s (CYP1B1, CYP2S1, CYP2U1, CYP3A5, andCYP51)were present at a significantly higher level of intensity compared with normal colon. P450 expressionwas also detected in lymph node metastasis and the presence of several P450s (CYP1B1, CYP2A/2B, CYP2F1, CYP4V2, and CYP39) in the lymph node metastasis strongly correlated with their presence in corresponding primary tumors. The presence of strong CYP51 (log-rank = 12.11, P = 0.0005) or strong CYP2S1 (log-rank = 6.72, P = 0.0095) immunoreactivity were associated with poor prognosis. CYP51was also an independent marker of prognosis (P = 0.009). Conclusions: The expressionof individual P450shasbeenestablished incolorectal cancer.Several P450s show increased expression in colorectal cancer. High expressionof CYP51or CYP2S1 were associatedwith poor prognosis and CYP51is an independent marker of prognosis. Colorectal cancer is one of the most common cancers in the Western world. The 5-year survival rate, although slowly improving, is still relatively poor at 40% (1). A significant proportion of patients present with locally advanced disease and current therapy for advanced colorectal cancer, which is based on a 5-fluorouracil regimen, results only in a modest improvement in survival (1, 2). Most large bowel cancers arise from adenomas and f5% of these adenomatous polyps progress to malignant tumors within 5 to 10 years (3). Environmental factors and genetic susceptibility both make important contributions to the development of colorectal cancer (3, 4). The cytochromes P450 (P450) are a multigene family of constitutive and inducible enzymes that have a central role in the oxidative metabolism of a wide range of xenobiotics and biologically active endogenous compounds (5, 6). The P450s are classified into families and subfamilies based on nucleic acid homology; there are currently 57 known human P450s. Some have been very well characterized, whereas little is known about the biology of the more recently identified P450s. Individual P450s show cell type– and tissue-specific patterns of expression (7, 8). The P450s have a major role in tumor development via their metabolism of many carcinogens (9). Compounds implicated in the etiology of colon cancer include polycyclic aromatic hydrocarbons and more especially heterocyclic amines, many of which require metabolic activation by P450s before exerting their genotoxic effect (10). Specific P450s have also been shown to be expressed in tumors; in particular, CYP1B1 is overexpressed in a range of tumors (7, 11, 12). Because the P450s are involved in the oxidative metabolism (activation and deactivation) of many anticancer drugs, they are capable of influencing the response of tumors to anticancer therapy (12, 13). The outcome in terms of activation (i.e., cytotoxicity) or deactivation (i.e., resistance) is determinant upon the relative amount and activity of specific P450s in individual tumor cells (7). Several therapeutic strategies are now being developed to exploit the presence, overexpression, and activity of P450s in tumors (14, 15). These approaches include P450 vaccines (16), P450-mediated prodrug activation (12, 17–19), and P450 inhibitors (20). The presence of CYP1B1 in tumors is currently being exploited as a tumor antigen. A phase 1 trial of a CYP1B1 www.aacrjournals.org Clin Cancer Res 2005;11(10) May1, 2005 3758 Authors’Affiliations: Department of Pathology, University of Aberdeen and Auvation, Ltd., Aberdeen, United Kingdom Received 9/10/04; revised1/4/05; accepted1/25/05. Grant support: The Health Foundation Awards, Teaching Company Scheme (Knowledge Transfer Partnership), and University of Aberdeen DevelopmentTrust. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Graeme I. Murray, Department of Pathology, University of Aberdeen, Foresterhill, AB25 2ZD, Aberdeen, United Kingdom. Phone: 44-1224553794; Fax: 44-1224-663002; E-mail: g.i.murray@abdn.ac.uk. F2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis Research. on May 1, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from DNA vaccine has been successfully completed (16) and a phase 2 trial of this vaccine will begin patient recruitment shortly. A number of prodrugs designed to be selectively activated by P450 enzymes are also currently being evaluated (12, 14, 17, 19). The bioreductive prodrug AQ4N, a topoisomerase inhibitor, is activated to the cytotoxic amine AQ4 by cytochrome P450–mediated bioreduction selectively under the hypoxic conditions found in tumor tissue. CYP3A4, CYP1A1, and CYP1B1 all contribute to bioreduction of AQ4N (17, 18). Several inhibitors of individual P450s are also www.aacrjournals.org Clin Cancer Res 2005;11(10) May1, 2005 3759 Table1. Details of the cytochrome P450 antibodies P450 antibody Source Type Immunogen Antigen retrieval* and antibody dilution for immunohistochemistry CYP1A1 Chemicon Polyclonal Peptide, amino acid sequence not stated in datasheet 20 min,1/1,000 CYP1B1 Own laboratory (23) Monoclonal Peptide, PENFDPARFLDKDGL (amino acids 437-451) 20min, undiluted tissue culture supernatant CYP2A6/2B6 Own laboratory Monoclonal C terminal peptide, RNYTMSFLPR (CYP2A6 sequence) 20min, undiluted tissue culture supernatant CYP2C8/9/19 Chemicon Polyclonal Peptide, amino acid sequence not stated in datasheet No antigen retrieval,1/500 CYP2D6 BDBioscience Monoclonal Expressed human CYP2D6 No antigen retrieval,1/20 CYP2E1 Oxford Biomedical Research Polyclonal Expressed human CYP2E1 20min,1/2,000 CYP2F1 Own laboratory Polyclonal COOH-terminal peptide, RPFQLCLRPR 20min,1/1,000 CYP2J2 Own laboratory Polyclonal COOH-terminal peptide, SHRLCAVPQV 20min,1/200 CYP2R1 Own laboratory Polyclonal COOH-terminal peptide, QPYLICAERR 20min,1/1,000 CYP2S1 Own laboratory Polyclonal COOH-terminal peptide, TDLHSTTQTR 20min,1/1,000 CYP2U1 Own laboratory Polyclonal COOH-terminal peptide, HPFNITISRR 20min,1/1,000 CYP3A4 Own laboratory (24) Monoclonal Purified human CYP3A4 20min, undiluted tissue culture supernatant CYP3A5 Own laboratory Monoclonal COOH-terminal peptide, DSRDGTLSGE 20min, undiluted tissue culture supernatant CYP3A7 Own laboratory Monoclonal COOH-terminal peptide, ESRDETVSGA 20min, undiluted tissue culture supernatant CYP3A43 Own laboratory Polyclonal COOH-terminal peptide, HLRDGITSGP 20min,1/1,000 CYP4F11 Own laboratory Monoclonal COOH-terminal peptide, RVEPLGANSQ 20min,1/10 CYP4V2 Own laboratory Polyclonal COOH-terminal peptide, KLKRRNADER 20min,1/1,000 CYP4X1 Own laboratory Polyclonal COOH-terminal peptide, NGMYLHLKKL 20min,1/1,000 CYP4Z1 Own laboratory Polyclonal COOH-terminal peptide, NGIHVFAKKV 20min,1/1,000 CYP24 Own laboratory Polyclonal COOH-terminal peptide, RELPIAFCQR 20min,1/1,000 CYP26A1 Own laboratory Monoclonal COOH-terminal peptide, PARFTHFHGE 20min,1/10 CYP39 Own laboratory Polyclonal COOH-terminal peptide QCRIEYKQRI 20min,1/1,000 CYP51 Own laboratory Polyclonal COOH-terminal peptide CPVIRYKRRSK 20min,1/1,000 *The antigen retrieval step consisted of microwaving the sections in 0.01mol/L citrate buffer (pH 6.0) for 20 minutes in an 800Wmicrowave oven operated at full power. Cytochrome P450 in Colorectal Cancer Research. on May 1, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from currently in development (20) and AVI-4557, an antisense construct specifically targeted against CYP3A4, has recently completed a phase 1 study (21). Gene-directed prodrug therapy is also being used to deliver exogenous P450s (21, 22). The presence of other P450s that may interact with prodrug activation would, therefore, have important clinical implications. In this study, we have conducted a comprehensive analysis of the expression of P450s in colorectal cancer and defined the expression profile of P450s in primary colorectal cancer, metastatic colorectal cancer, and normal colon. We have identified P450s that are overexpressed in colorectal cancer and those associated with poor prognosis. Materials andMethods Antibodies. A panel (n = 23) of P450 antibodies was used in this study. The development of monoclonal antibodies to CYP1B1 (23) and CYP3A4 (24) has been described previously. Polyclonal antibodies to the following P450s—CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP3A43, CYP4V2, CYP4X1, CYP24, CYP39, and CYP51—were produced by immunizing rabbits with the relevant COOH-terminal peptide (Table 1) conjugated to ovalbumin. The use of COOH-terminal peptides as immunogens has been previously used to successfully develop antibodies to individual P450 forms (25). Animals received two booster immunizations at 4to 6-week intervals after the initial immunization. Animals were bled 7 to 10 days after the last injection and serum obtained by centrifugation of the clotted blood. Monoclonal antibodies to CYP2A6, CYP3A5, CYP3A7, CYP4F11, and CYP26A1 were produced as previously described (23, 24). In each case, the appropriate COOHterminal peptide conjugated to ovalbumin was the immuno