Cytochrome P450 2W1 (CYP2W1) as a novel drug target in colon cancer therapy

Detta är en avhandling från Stockholm : Karolinska Institutet, Dept of Physiology and Pharmacology

Sammanfattning: Cytochrome P450 2W1 (CYP2W1), is a monooxygenase enzyme endogenously expressed in fetal colon and normally silenced after birth. In adult life, the expression of CYP2W1 occurs exclusively in tumor cells yielding high amounts of the enzyme in 30% of human colon cancers. Remaining the third most commonly diagnosed malignancy in the world, the colon cancer requires new chemotherapeutic strategies that would provide higher s electivity towards transformed cells. The unique confinement of CYP2W1 to tumor tissue opens the possibility for its targeting in colon cancer therapy. The studies in this thesis have focused on characterization of the CYP2W1 enzyme and the development of novel anticancer prodrugs designed for CYP2W1 - specific activation. In cancer tissues and transfected cells CYP2W1 gives multiple immunoreactive bands, suggesting that the protein might be subjected to posttranslational modifications. Our in vitro and in vi vo studies reveal that CYP2W1 undergoes glycosylation at Asn177, which is enabled by the unique inverted topology of the protein in the ER membrane. Regardless of the reversed emplacement, CYP2W1 retains its catalytic function in cell systems and is capabl e of converting inactive substrates to potent cytotoxic species, demonstrating the potential for prodrug activation. With the goal to develop antitumor prodrugs for specific activation by CYP2W1, we have employed a library of novel duocarmycin - based compo unds and identified ICT2705 and ICT2706 as the first molecules whose cytotoxic activity is dependent on CYP2W1 enzyme. Our studies reveal the results of the first attempt of CYP2W1 targeting in cancer, both in vitro and in vivo . We show that the colon canc er cell lines expressing CYP2W1 suffer substantial DNA damage and rapid loss of viability following incubation with ICT2705 and ICT2706. Moreover, we demonstrate that the CYP2W1 - positive human xenografts undergo complete growth inhibition in mice dosed wit h ICT2706, with no apparent deleterious effects detected in any of the treated animals. In addition, we present in vitro evidence that the potent cytotoxic effect is most likely propagated by a bystander killing mechanism. Polymorphisms in CYP2W1 gene yiel d two alleles of significant frequencies, designated CYP2W1'2 and CYP2W1'6. The resulting variant enzymes carry missense mutations that might affect the therapeutic outcome of prodrug targeting as well as the course of the metastatic disease. A recent stud y has associated CYP2W1'2 with a decreased risk for colon cancer, suggesting the altered catalytic activity of this enzyme variant. Using the novel duocarmycin substrates, we were able to evaluate the catalytical properties of the variant enzymes for the f irst time. In contrast to previous findings, our studies show comparable catalytic capacity for all CYP2W1 enzymes, and no association between CYP2W1'2 genotype and colon cancer risk based on 10 - fold larger patient cohorts. In conclusion, we have found tha t CYP2W1 is a glycosylated enzyme with reversed ER topology, whose catalytic activity is sustained in an intact cell system and unaffected by the most frequent polymorphic changes. We have developed first prodrugs for CYP2W1 targeting in cancer and demonst rated their antitumor efficacy in a preclinical setting. The work presented herein provides the basis for a novel therapeutic approach in colon cancer chemotherapy.

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