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NOTE TO USERS This reproduction is the best copy available. ® UMI Proprotein Convertase 1 and 2 Profiles in Human Liver Colorectal Metastases. George N. Tzimas, MD L.D. MacLean Surgical Research Laboratories Department of Surgery Division of Experimental Surgery The Royal Victoria Hospital McGill University, Montreal January 2004 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master ofScience in Experimental Surgery Copyright © George N. Tzimas Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 0-612-98754-X Our file Notre référence ISBN: 0-612-98754-X NOTICE: AVIS: The author has granted a non L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada TABLE OF CONTENTS Page 1. Abstract 3 II. Resume 4 III. Introduction 5 IV. Background 1. Proprotein Convertases 7 2. PCs in Cancer and Clinical Relevance Il 3. Purpose of the study 14 V. Materials 15 VI. Methods 15 VII. Results 21 VIII. Discussion 25 IX. Acknow ledgements 31 X. Figure Legends 32 XI. References 40-64 XII. Figures XIII. Appendix 2 ABSTRACT The family of proprotein convertases has been recently implicated in tumorigenesis and metastasis in animal models. However, these studies have not yet been completely corroborated in human tumors. Here, we show that mRNA, prote in expression, and prote in cIeavage profiles of proprotein convertases 1 and 2 are altered in Iiver colorectal metastasis, compared to unaffected and normal liver. Active PC 1 is overexpressed in tumor, correlating with its mRNA profile. Moreover, the enhanced PC2 processing pattern in tumor correlates with the overexpression of its specifie chaperone 7B2, which in turn may represent a target for early diagnosis and treatment. The increased PC2 maturation, as weIl as the overexpression and altered processing of PC 1 may be either a cause or a consequence for the observed metastatic phenotype. Nevertheless, they may result in the alteration of the secretory pathway, which could therefore, modify the cellular microenvironment and thus favor tumor growth and/or metastasis. 3 RESUME La famille des proprotein convertases est impliquée dans la formation de tumeurs et métastases chez les modèles animaux. Cependant, ces études n'ont pas encore été complètement corroborées chez les humains. Ici, nous prouvons que la mRNA, l'expression des protéines, et les profils de segmentation de convertases 1 et 2 sont changés dans les métastases colorectales du foie comparé au foie normal. PC 1 actif est abondant dans la tumeur, se corrélant avec son profil de mRNA. D'ailleurs, le traitement du modèle PC2 qui est accru dans la tumeur se corrèle avec l'abondance de son chaperon spécifique 7B2, qui peut représenter une cible pour le diagnostic et le traitement. La maturation du PC2 accrue, comme l'abondance et la nature changée de PC 1 pourrait être une cause ou une conséquence du phénotype métastatique. Néanmoins, elles peuvent avoir comme conséquence le changement des routes de secrétions, modifiant ainsi le micro-environnement cellulaire et favorisant ainsi la croissance et/ou la formation de métastases des tumeurs. 4 Introduction Liver metastasis from colorectal primary cancer remains the most common reason of therapeutic failure after successful surgi cal intervention (colectomy). 1t has been estimated that approximately 300.000 patients are given the diagnosis of colorectal cancer (CRC) in North America and Europe every year(l, 2). From all these patients, 60% will eventually develop hepatic metastases. Without aggressive surgical and medical treatment, 5-year survival among this group is rare. Even with surgery and aggressive chemotherapy the 2-year survival is approximately 85% and declines further on (3, 4). The main theme of "seed-soil" is not enough to explain the preferential metastatic potential of CRC to the liver, based on the differences in venous and lymphatic drainage of the se intra-abdominal organs. Therefore, the fact that the colon is drained via the tributaries of the portal vein, finally entering into the liver most likely is not the only reason for this observation. Recently the basis of this preferential metastatic potential of other types of tumor, such as breast carcinoma and renal metastases, has been described in the literature. Several proteins involved in this phenomenon, such as the chemokines have been implicated in this observed bias (5-7). Furthermore, in recent years, the role of the proprotein convertase family (PCs), a series of enzymes responsible for "transforming" proteins from their pro-active to their active isoform, in several states of disease, including carcinogenesis and metastasis has been clearly demonstrated (8-11). 5 Finally, it has been postulated that neuroendocrine differentiation profile is associated with worse outcome in colon cancer. The purpose of this study was to evaluate the presence and the expression pattern oftwo specific proprotein convertases, PCI and PC2, in colorectal liver metastases and to compare this profile with the pattern observed in normalliver specimens. We also assessed the presence and expression of 7B2, a chaperone that facilitates the processing of PC2 within the same samples. 6 Background Pro protein Convertases To regulate biological activity, a wide variety of proteins is synthesized as inactive precursors within the secretory pathway. Over the last thirty years our understanding of the complex cellular machinery involved in the processing of inactive secretory precursors into active polypeptides and proteins by limited proteolysis has greatly expanded. It is clear now that following the removal of the signal peptide; precursor cleavage can occur either intracellularly, at the cell surface, or within the extracellular milieu. The evolution ofthis mechanism involves several enzymes that are tightly controlled in a temporal and spatial fashion. These processing enzymes usually cleave proproteins at selected sites composed of single or paired basic amino acids. It is clear thus, that the generation of active proteins and oligopeptides requires the precursor polypeptide, which acts as a substrate and the proteolytic enzyme(s) responsible for the conversion of the precursor into its final biologically active protein/peptide product. The old debate about the presence of unique processing enzymes for each precursor, or of enzymes that albeit few in number can process several precursors and have redundant functions, has been recently brought to sharp focus and answered. The secretory enzymes responsible for the intracellular processing of the precursors have been identified and functionally 7 characterized. These "Precursor Convertases" (PCs) belong to a phylogenetically conserved family of subtilisin-like (subtilases), calcium dependent serine proteinases (Figure 1). The homology of the structure of their catalytic domain, places them in the family of the kexin subfamily of subtilases. Their structural motif includes a signal peptide followed by pro, catalytic, middle and cytoplasmic domains. To date eight members ofthis family have been identified, namely furin, PClIPC3, PC2, PC4, PACE4, PC5IPC6, PC7/LPCIPC8 and SKI-lISlP (12-46). Recently, an additional member of the convertase family has been described NARC-l, but its role is still under investigation (47, 48). The PCs are usually activating proteases, and have not been reported to inactivate polypeptides, a process usually performed by degradative enzymes. Furin, PCl, PC2, PC5, PACE, PACE4 and PC7 cleave precursor proteins at basic residues within the general motif (ArglLys) (X)n - Arg -..v where n= O.2.4.or 6 and X is any amino acid except Cys and rarely Pro. In contrast, SKI-l cleaves precursors at non-basic amino acids within the motif (RlK.)-X-(L,V)-(L,T,K,F). Among these enzymes only Furin, PC5, PC7 and SKI-l possess a transmembrane

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