Colon cancer specific cytochrome P450 2W1 : polymorphism, membrane topology and endogenous roles in development

Sammanfattning: Cytochrome P450 2W1 (CYP2W1) belongs to a family of drug metabolizing monooxygenases with an unidentified yet endogenous function. CYP2W1 expression pattern has characteristic features of oncofetal genes. Previous studies have shown significant levels of CYP2W1 protein in 30% of human colon cancers and in 50% of corresponding liver metastases. High expression of CYP2W1 is an independent prognostic factor for poor survival outcomes in colorectal cancer (CRC) patients. Such tumor specific feature renders CYP2W1 an attractive target for colon cancer therapy. Indeed, CYP2W1-mediated activation of a number of duocarmycin related chloromethylindolines demonstrated strong and selective cytotoxic effects toward cancer cells in vitro and in vivo. CYP2W1 is a polymorphic gene. The most frequent genetic variants, are CYP2W1'2 and CYP2W1'6 that carry missense mutations producing amino acid changes that might affect the enzyme function. A recent study suggested that CYP2W1'2 variant is associated with decreased risk of colon cancer. We have analyzed a 10-fold larger patient cohort where no such differences in the distribution of CYP2W1'2 and CYP2W1'6 between CRC patients and healthy controls could be detected. Moreover, the functional analysis of these enzyme variants demonstrated equally potent capacity to metabolize the chloromethylindoline substrates. CYP2W1 enzyme is glycosylated at Asn177 in cancer tissues and in transfected cells, suggesting that in contrast to other P450s it has an inverted endoplasmic reticulum (ER) membrane topology with the bulk of the protein facing the lumen. We now confirmed such membrane orientation based on a number of assays using FLAG-tagged CYP2W1 and also CYP2W1 fused with a redox sensitive luciferase reporter that displays full activity only upon its oxidative folding in the ER. Such ER topology apparently should hinder CYP2W1 interaction with its canonical cytosol oriented redox partners, cytochrome P450 reductase (POR) and/or cytochrome b5 reductase. This was confirmed by unaffected functional activity of CYP2W1 under the conditions of knocked down/inhibited POR and cytochrome b5 reductase. Interestingly, the nonglycosylated Asn177Ala mutant displayed significantly decreased catalytic activity. This study strongly supports the atypical membrane topology of CYP2W1 and suggests the interaction of CYP2W1 with a hypothetical ER luminal electron donor. CYP2W1 is transiently expressed during gut development. We described a developmental expression profile of CYP2W1 in murine and human gastrointestinal tissues. The gene expression is initiated at the early stages of gestation and is completely silenced shortly after birth. Such fetal specific expression was shown to be epigenetically regulated and indicates possible endogenous role in gut development. Cyp2w1 knock out (KO) mouse model was established in order to investigate such possibility. Phenotype analysis revealed decreased crypt length in the distal colon epithelium of adult Cyp2w1 KO mice whereas the transcriptomic profiling identified a number of genes being down-regulated. The most affected genes were found to be involved in the cell adhesion and extracellular matrix establishment. In conclusion, we have characterized polymorphic variants of CYP2W1 and found no significant correlation between their distribution and CRC risk. The completely inverted membrane topology of catalytically active CYP2W1 suggests its interaction with redox partner yet to be identified in the ER lumen. The explicitly developmental pattern of expression, the phenotypic and gene expression changes in Cyp2w1 KO gut tissues indicate a potential role of CYP2W1 in gut development.