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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

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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 domain and circulate

between the Trans Golgi Network (TGN) and the cell surface. The above

enzymes as weIl as PC4, PACE 4 and PC4 are involved in the cleavage of

proteins of the constitutive pathway. This is in contradiction with PC 1 and

PC2 that process proteins of the secretory pathway and are located in the

dense core secretory granules. These two PCs are produced in the

Endoplasmic Reticulum (ER) as pro active precursor proteins, are transported

8 through the Golgi apparatus and the TGN into the secretory granules and are proteolytically cleaved along the way.

So far, the only known substrates for the enzyme SKI-I are: pro-brain derived

neurotrophic factor, sterol regulatory element-binding proteins, the

endoplasmic Reticulum-stress response transcription factor A TF6, and the

surface glycoprotein GP-C of Lassa virus. Thus, PCs are responsible for

processing of neuropeptides, receptors, growth factors, cell surface

glycoproteins, and enzymes, whereas SKI-I cleaves proproteins that control

cholesterol and lipid metabolism and are involved in neural protection and

growth and in the endoplasmic Reticulum-stress response pathway.

After proteolysis by the convertases, usually the mature proteins/peptides are

subject to several other modifications necessary to achieve full biological

activity. The most common being the removal of carboxy-terminal basic

residues by carboxypeptidase E or D.

Commonly to other proteases, PCs are initially synthesized as inactive

proproteins with aN-terminal pro segment extension. Autocatalytic cleavage

of this region leads to activation of the PCs.

In addition, a well-described oligopeptide, 7B2 acts as a chaperone for PC2 by

preventing immature activation of the zymogen. 7B2, which is produced in

the ER as a pro-7B2 peptide is attached to the pro-PC2 in the ER facilitated by

the relative alkaline conditions of this compartment. This inactive complex is

transferred through the TGN where pro-7B2 is cleaved in a C-terminal peptide

and an N-terminal protein. Pro-PC2 then is autocatalytically cleaved after the

9 prodomain while the complex is transported in the secretory granules. In the acidic environment of these organelles, the prodomain and the 7B2 fragments dissociate from the enzyme, which then becomes fully active. Proper folding of PC2 is mandatory for the attachment of 7B2. Although 7B2 facilitates appropriate processing ofpro-PC2, leading to the active form, it has been

shown that the carboxy 1 region of the pro-7B2 may block further pro-PC2 maturation. It is believed, that pro-7B2 cleavage is required for pro-PC2 cleavage (23, 49-54).

Furthermore, a recently discovered, specifie and potent PC 1 inhibitor was recently described. ProSAAS is a regulatory peptide that has been shown to

exhibit inhibitory effects on PCI (55-59). It is interesting to note that while

ProSAAS and 7B2 are not homologous, they are of similar sizes, with a N­ terminal proline-rich region, both contain several pairs of basic amino acids

and are abundantly expressed in endocrine and neural tissues. The cleavage profile of ProSAAS was recently reported to be in a tissue-specifie fashion at

its C-terminus into smaller inhibitory peptides. Furthermore, it has been reported that the 66 kDa isoform of PC 1 is better inhibited than the 87 kDa precursor form (56, 58, 59).

10 PCs in Cancer and Clinical Relevance

The PCs have been recently implicated in the process of tumorigenesis as weIl as metastasis. lndeed, multiple cellular processes, i.e. suppression of gene expression or enzyme inhibition, support the hypothesis that PCs play a role in the genesis and progression of different proliferative disorders, including cancer. Although elevated expression of several PCs has been reported for different human cancers as weIl as for different tumor celllines, the relative

importance of various PCs in these cancers has not yet been clarified (1-6, 8-

Il, 14, 60-92). Tumor expression ofPCs can be studied at the prote in level by

techniques such as immunohistochemical staining and Western Blot analysis,

and at the mRNA level by reverse-Transcriptase Polymerase chain reaction,

Northern blot analysis, Rnase protection, or in situ hybridization.

Early studies revealed a high furin expression in advanced lung tumors (11,

82). That association has been recently confirmed in other malignancies such

as breast (10, 73) and he ad and neck (62, 75, 77) cancers. Based on the above

studies furin expression in tumors may constitute a significant prognostic

factor independent of other clinicopathological features of the tumor. Other

studies have confirmed the significant association between high expression of

PC 1 and PC2 in neuroendocrine tumors, suggesting thus their involvement in

the malignancy oftumor cells with a neural or endocrine phenotype (11, 69,

93-95). Although the studies have shown a positive association between PCI

Il and PC2 expression and the extent oftumors, further research is needed to elucidate the importance of these convertases in these endocrine-related

cancers.

Studies involving the prognostic significance of PACE4 expression in tumors

are less conclusive. The expression ofPACE4 has been reported to be higher

in breast cancer (10). In the same line PACE4 expression has been found to be

elevated in human he ad and neck malignancies and tumor celllines (9, 70). In

addition, using an animal model of human squamous cell carcinoma the previous group has shown that PACE4 expression is implicated in the process

oftumor progression and invasiveness (70).

The role ofPC5 and PC7 has not been examined than in few tumors. These

studies have shown a positive association between PC7 expression and the

extent ofbreast tumors ofwhich PC5 was not detected. Nevertheless, the

clinical relevance ofthese observations is not known yet.

Finally, the role of SKI-l and NARC-l is still under investigation, so their

biological role in disease states such as cancer in not yet established.

The exact role of the modulation of PC expression and/or activity in tumor

development and metastasis remains unclear. Nevertheless, because PCs are

directly responsible for the activation of cri tic al proteins implicated in

neoplasia, they may be targets of cancer therapy. Among the PC substrates,

matrix metalloproteinases (MMP), growth factors and adhesion molecules

through degradation of the extracellular matrix, modulation of cell growth

and/or migration are involved in tumor progression and metastasis.

12 Indeed, stromelysin-3, a member of the MMP family, ofwhich expression has been correlated in the past with local aggressiveness and poor clinical outcome, is cleaved to its active biological form by furin before secretion to the extracellular milieu (88, 90). ln the same line, MT-MMPs, a new family ofMMPs, have been shown to be overexpressed in colon and brain cancers and possibly to be involved in metastasis formation. Pro-MMP-2, a collagen IV degradation enzyme is cleaved by MTI-MMP. The last enzyme possesses 2 possible cleavage sites by furin (9, 62, 65, 70, 75, 79, 96-100).

Another example of such a biological activity is the family of the adamalysin metalloproteinases (ADAMs). These proteins, when active, have been shown to be involved in a variety of activities such as: fertilization, myogenesis, neurogenesis and shedding of other proteins. Within these proteins, cytokines and growth factors, such as TGF-a, TNF-a, EGF, CSF-I, TNFR2 and FAS-L are known to be involved in carcinogenesis and metastasis. The ADAMs have been shown as weIl to be cleaved to their active moiety by furin like eonvertases (80).

Cell adhesion molecules are cell surface proteins that control cellular traffie, transmigration through the endothelium, homing in and localization to various target organs during inflammation and tumor cell colonization. Finally, several ofthese moleeules, sueh as members of the immunoglobulin, the integrin and the selectin family have been shown to be cleaved by convertases

(84, 97, 98, 101-108).

13 It is obvious thus, that PCs are directly or indirectly involved with tumorigenesis and metastasis formation.

Pnrpose of the stndy

In this study, we elected to assess the expression profile, at a gene and at a

prote in level of PCI and PC2. The main reason is that the se two PCs are

implicated in the secretory pathway. Indeed, it is well known that the colonic

mucosa contains a large amount of neuroendocrine cells. It would make sense

to have a similar cell population at areas of colon carcinoma and furthermore

at the areas of liver metastasis. In addition, previous reports have documented

the immunohistochemical localization of PC 1 and PC2 in the human anal

canal (109). PC 1 and PC2 also have been implicated in the processing of pro­

neurotensin, a well known colonic epithelium growth factor( 45, 54, 110-113),

that might be involved in colonic carcinogenesis.

Finally, it has been also postulated that colon cancers possessing a

neuroendocrine profile by immunohistochemistry, behave in a more clinically

aggressive fashion (66, 91, 114-120).

We believe thus, that there might be a specific and different profile of these

two neuroendocrine PCs (PC 1 and PC2) within the liver metastases, compared

to normalliver. We furthermore tried to assess the presence and the profile of

the PC2 chaperone, 7B2 at the gene and prote in level within the same

samples.

14 Materials and Methods

Human specimens

Tumor specimens (T) as weil as their unaffected liver (UL) counterparts were collected from 14 patients that underwent liver resection for CRC metastases at the McGill University Healthcare Center. Normalliver specimens (N) were obtained from previously healthy organ donors. Ail the specimens were harvested immediately after resection at the Pathology Suite, and were snap frozen in liquid nitrogen. 48 hrs later, ail the specimens were stored in-80°C degrees.

Ail the specimens were collected with appropriate informed consent, and according to the policies of the Institutional Review Board (see appendix).

Preparation of RNA

Total RNA was isolated from the samples using the TRIzol technique. TRIzol reagent was obtained from life Technologies and Chloroform was provided by

EM Science. Isopropanol was provided by Sigma Chemical and 75% ethanol was prepared by diluting 100% ethanol from Commercial Alcohols (Montreal,

PQ) and DEPC, RNAse free water. Homogenized tissue in 1 mL of TRIzol

15 was subjected to phase separation by adding 0.2 ml chloroform per 1 ml

TRIzol, shaking vigorously, incubating for 2-3 minutes at room tempe rature and then centrifuging at 12,000 x g for 15 minutes at 4°C. The clear aqueous phase was collected while the chloroform/non-aqueous phase and interphase were discarded. RNA was precipitated by adding 0.5ml isopropanol for each

1ml TRIzol originally used. The samples were incubated at room temperature for 10 minutes followed by centrifugation for 10 minutes at 12,000g at 4°C.

The supematant was subsequently removed and the pellet was washed with

1ml 75% ethanol by centrifugation at 7500g for 5 minutes at 4°C. The washed pellet was resuspended in DEPC water.

Determination of RNA Concentration

2fll of extracted RNA was suspended in 500fll of DEPC water and optical

densities were measured using a spectrophotometer. From the measurement of

optical density at 260 nm, the concentration of RNA was calculated using the

equation (optical density value)(0.04flg)(250) to give a value in flg/fll.

Protein extraction

For prote in extraction, approximately 0.5 mg of sample tissues were

homogenized using the Polytron apparatus, as previously described (121).

TX-100 was added to a 1% final concentration and tissue extract solubilized

16 for 30' at 4°C. Lysates were then centrifuged at 10000 g for 2 x 30' at 4°C.

Prote in concentrations in the supernatants were determined using the Bradford

method.

PCR prim ers and Antibodies

PCI primers were designed as follows f5'-

TGGCTTGCTAAA TGCCAAAGCTC-3' /r5'­

ATCCACCATCTTCTCCACCCC-3', PC2 primers were f5'-

GTCCTTGATGCAGGTGCCATC-3'/r5'­

ACTCCTTCAGCACCCCCTTC-3' .

Finally, 7B2 prim ers were fS'-CACCAGGCCATGAATCTT-3'/r5'-

CTGGATCCTTATCCTCATCTG-3'. We used GAPDH as the

housekeeping gene, allowing us to measure optic density ratios in 1% agarose

gels and consequently to perform a semiquantitative analysis of the numbers

of copIes. The GAPDH primers were the following f5'­

CCCTTCATTGACCTCAACTACATGGT-3' /r5'­

GAGGGGCCATCCACAGTCTTCTG-3' .

For the immunoblots, we used rabbit antisera raised either against PCI (amino

acids 621-726, Figure 3), PC2 (amino acids 529-637, Figure 4) or 7B2 (amino

acids 32-39, Figure 5). AlI these antibodies were generously given by Dr.

Nabil Seidah, and their use has been previously published in the literature (23,

109).

17 Reverse Transcriptase Polymerase chain reaction (RT-peRl

For RT-PCR, 4ug of total RNA were reverse transcribed using oligo (dT) primers and Thermoscript® Reverse Transcriptase (Life Technologies). PCR amplification was performed with Platinum® Taq Polymerase (Life

Technologies) and commercially synthesized specifie primer pairs (BioCorp

Inc. Montreal, PQ). The PCR reaction was carried out using 10X PCR Buffer,

50mM MgCh, 10mM dNTP mix, 40 pmol from each primer and 0.5 Units of polymerase per reaction. Nucleotide sequences for PCR primers were previously described. Amplification programs were as follows: PC 1 was

amplified with the sense and the antisense yielding a 553 bp cDNA by one

cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 30 seconds 60°C

for 30 seconds and noc for 30 seconds, and a final cycle of noc for 7 min.

PC2 was amplified with the sense and the antisense yielding a 422 bp cDNA

by one cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 30 seconds

60°C for 30 seconds and noc for 30 seconds, and a final cycle of noc for 7

min.

7B2 amplification was performed with the sense and the antisense primer by

one cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 30 seconds

60°C for 30 seconds and noc for 30 seconds, and a final cycle of noc for 7

min.

The amplification of GAPDH was performed us mg the sense and the

antisense primer, yielding a 470 bp cDNA by one cycle at 94°C for 5 min,

18 followed by 27 cycles at 94°C for 30 seconds 60°C for 30 seconds and noc for 30 seconds, and a final cycle of noc for 7 min.

PCR products were separated by electrophoresis on 1% TAE-agarose gel,

stained with ethidium bromide, and photographed under UV using the

Chemimager® system. Optic densities were calculated by the same system

and for every specimen an optic density ratio between the gene of interest and

the respective GAPDH OD was measured, allowing thus semi quantitative

analysis of the cDNA amplification products.

Immunoblot

For immunoblots, identical amounts of total prote in (100 ug per lane) were

subjected to electrophoresis in 7% -12% SDSIPAGE and blotted onto a

nitrocellulose membrane. Membranes were blocked with 5% milk and then

immunoprobed at 4°C overnight with anti-PC1 (l: 500), anti-PC2 (1: 500) and

anti-7B2 (l :500). The concentration used for each antibody was titrated to the

best results. Then the membranes were incubated with Prote in A-Horseradish

Peroxidase Conjugate (Bio-Rad, Richmond CA) at a concentration of 1:5000.

An enhanced chemiluminescence detection system (Amersham Pharmacia)

was used for band detection. We used a semiquantitative analysis, based on

the optic density ratios of the different isoforms of the proteins or

oligopeptides detected.

19 Immunohistochemistry

4 f..lm- thick, formalin-fixed, paraffin-embedded tissue sections, were treated as previously described (109). The slides were incubated with the same anti­

PC 1, PC2 and 7B2 antibodies at a 1 :2000 dilution.

Following treatment the sections were revealed with biotinylated goat-anti­ rab bit IgG-B (1 :200), combined with Histocain-plus kit, followed by 3-amino-

9-ethylcarbazole (AEC) chromogen and counterstaining with hematoxylin.

All the slides were assessed by an experienced immunopathologist as weIl as by an experienced biologist.

Statistical analysis

Statistical analysis of the data was performed using the statistical software

Systat, version 9.01 (1998, SPSS Inc, Cary, NC). Univariate analysis of continuous data was compared by two-tailed unpaired t-test. Differences in proportions were compared by univariate Chi-squared analysis. P values less than 0.05 were considered statisticaIly significant.

20 Results

A. PCl and PC2 mRNA are expressed in both normal and unaffected liver but their expression is differentially regulated in tumor

We initiaUy assessed the presence of the mRNA encoding PC 1 and PC2 in liver CRC metastatic tumors versus unaffected and normallivers.

We found PC 1 and PC2 mRNA to be present in aU the specimens.

Specifically, PCI and PC2 RT-PCR amplification products were of the expected size, 553 bp and 422 bp respectively (Figure 2A).

PCI mRNA expression was two-fold higher in areas of tumor compared to areas of unaffected and normalliver (P

In contrast, PC2 mRNA was overexpressed two-fold in areas of unaffected and normalliver compared to areas of metastasis (P

B. PCl protein expression and maturation is altered in tumor

To correlate mRNA and prote in expression, we examined total sample lysates by immunoblotting. Relative amounts of prote in were quantified by scanning densitometry of the immunoblots.

Interestingly, the anti-PCI antibody (Fig. 3A) revealed two immunoreactive species of 84 and 66 kDa, respectively (Fig. 3B), in tumor (T), unaffected (U), and normal (N) liver. The higher molecular mass isoform (84 kDa)

21 corresponded to the full-length previously described active PCl, whereas the

C-terminally immunoreactive 66 kDa isoform was likely to be an N­ terminally truncated, inactive form ofPCI (Fig. 3A) (14,15). ln tumors we found the total amount ofPCI (p84+p66) to be -2.5-fold elevated compared to unaffected and normal samples (Fig. 3C, left panel).

Moreover the ratio of the 84 kDa active form over the 66 kDa form was also

-2.5 times higher than in both unaffected and normalliver (Fig. 3C, right panel).

c. pe2 protein expression and maturation is altered in tumor

Immunoblot analysis with anti-PC2 antibody (Fig. 4A) revealed two species of75 and 66 kDa respectively, representing the inactive proPC2 isoform and the cleaved, active PC2 isoform (Fig. 4B) (14). ln contrast to PCI, the relative amount of total PC2 (p75+p66) was higher in unaffected and normalliver than in metastasis (Figure 4C, left panel).

Nevertheless, a similar pattern ofprocessing was observed, i.e. in metastasis the fully active 66 kDa form predominates over the inactive 75 kDa pro-form, while in unaffected and normalliver the proPC2 isoform was more abundant.

Again the PC2 processing ratio of active PC2 (P66) over the inactive proPC2

(p75) in tumor versus unaffected and normalliver indicated a -1 O-fold increase in PC2 maturation in liver metastasis (Figure 4C, right panel). In

22 conclusion, alteration of PC 1 and PC2 processing, le ad to increased accumulation of active isoforms of both convertases in liver metastasis.

D. The PC2 chaperone, 7B2, is expressed only in Iiver CRC

metastases.

Since PC2 processing is controlled by its specific binding protein 7B2, we

quantified 7B2 mRNA expression profiles in normal and unaffected livers

compared to liver metastasis.

RT-PCR amplification of7B2led to an expected 454 bp product (Fig. 5A),

and showed a ~3-fold (P

compared to unaffected or normalliver (Figure 5B).

By immunoblotting, we were not able to detect any 7B2 in both unaffected

and normalliver, while an intense immunoreactive band was observed at the

expected 7B2 molecular mass of21 kDa (8) in metastasis samples (Fig. 6B).

E. Immunohistochemical analysis of PCl, PC2, and 7B2 in Iiver

metastases, unaffected Iiver and normalliver, corroborates the previous

results.

U sing the antibodies described above, we characterized PC 1, PC2 and 7B2

localization by immunohistochemistry.

23 PC 1 staining was cytoplasmic and more abundant in areas of tumor than in adjacent unaffected liver, where only a few cells were positively stained (Fig.

SA,B,C), thus correlating with the mRNA and immunoblot data.

PC2 was present in areas oftumor, and adjacent unaffected parenchyma (Fig.

lOA,B, C).

Finally, 7B2 was detected by very strong cytoplasmic staining in areas of liver metastasis while adjacent unaffected liver did not stain (Fig. 12A,B, C).

In addition, although normalliver stained for PC 1 and PC2, 7B2 remained undetectable (Fig. 7,9,11).

24 Discussion

In this study, we assessed the presence and the cleavage/processing patterns of the two major convertases of the regulated secretory pathway, PCI and PC2,

as weIl as the PC2 chaperone 7B2. This study was performed in hum an liver metastases specimens from colorectal primaries and in unaffected liver

samples from the same patients that underwent liver resection, as weIl as in normallivers.

To our knowledge, this is the first report describing the expression ofPCI,

PC2 and 7B2 in human liver tissues. More importantly, we noted several

important points:

(i) At the mRNA level, PC 1 is overexpressed in tumor versus unaffected

and normalliver, while PC2 expression is downregulated (Fig. 2);

(ii) Consistently with the mRNA data, at the prote in level, the total

amount ofPCI (p84+p66) is overexpressed (Fig. 3C), while PC2

(p75+p66) is downregulated in tumor (Fig. 4C);

(iii) Both active PC 1 and PC2 isoforms are predominant in the tumor

samples (Fig. 3C, 4C);

(iv) In accordance with the enhanced PC2 zymogen-processing pattern, in

tumor, 7B2 mRNA is overexpressed (Fig. 5B);

(v) While 7B2 mRNA is present in aIl the samples, we were unable to

detect 7B2 by immunoblot in normal or unaffected liver samples

(Fig.6).

25 (vi) AIl the above results were corroborated by immunohistochemistry

analysis (Fig. 7,8,9, 10,11,12).

Several conclusions can be drawn by these results. These data support a negative feedback mechanism regulating PC2 mRNA expression, when PC2 proteolytic activity is overwhelming. Indeed, in tumor, abundant active PC2 may lead to a downregulation of its mRNA, and vice versa in unaffected and normalliver. Furtherrnore, the PC2 prote in profile aIlows us to support the hypothesis of a tightly regulated active PC2 production in the tumor, with

7B2 playing a leading role in this mechanism. Therefore, the unique in the tumor prote in detection of 7B2, as weil as its upregulated mRNA expression in the same samples, leads to an enhanced cleavage of PC2, from its inactive

(P75) to its active isoforrn (P66).

It is of no surprise also the observation that, in CRC metastases, the relative total amount ofPCl was ~2.5-fold that found in normal or unaffected liver, but also that the ratio of active to inactive PC 1 isoform (p84/p66) is also

~2.5-fold more in CRC metastases compared to their normal or unaffected counterparts. The explanation lies behind the fact that PC 1 is being auto cleaved (122). Therefore, an x-fold increase (in tumor) of the total amount of the PCI zymogen should lead to a similar x-fold increase (in the same specimens) of its active isoform (P84).

Interestingly, PC 1, pe2 and 7B2 are considered as markers of endocrine and neuroendocrine phenotypes (17,28,52,54,109, 121, 123-147). The fact that

26 they are also detected in human colon (13) from which the primary cancers and eventually metastases develop suggests that a neuroendocrine differentiation pro gram could take place during colon carcinogenesis and liver metastasis.

In addition, neuroendocrine differentiation profile has been suggested as a poor prognostic feature for primary colorectal cancer by several authors (91,

114-116, 148). The fact that the same "neuroendocrine" profile is observed in the liver metastases, can lead us to the hypothesis that this profile could be predictive of a "metastatic" potential of a specific subclone from the primary tumor (Fig.14).

Recently, a specific REl-lk silencer element in the promoter ofPC2 was identified (17) and binding oftranscription-silencing factors to this element may contribute to repression of the PC2 gene in non-neuroendocrine cells. It would thus be interesting to evaluate the level of such silencer in colorectal tumors and in their metastatic progeny.

Our results are in agreement with recent experimental animal evidence highlighting the potential implication of convertases in tumorigenesis and metastasis (8-11, 62, 64, 73, 75, 77, 83, 93). Indeed, Khatib et al. have shown that convertase inhibition in the HT-29 colon cancer cell line is followed by decreased invasiveness and tumorigenicity (8). Using al-PDX (a non-specific

PCs inhibitor) transfected HT-29 colon cancer cell-lines, they were able to demonstrate a reduced invasiveness that paralleled decreased mRNA levels of: urokinase-type plasminogen activator, receptor for the tissue-type

27 plasminogen activator and plasminogen activator inhibitor-1. Ali the above

proteins are normally processed by PCs.

In addition it has been shown that furin, another member of the convertase

family, is implicated in tumor progression in human head and neck

malignancies, with more aggressive cancers overexpressing different members

of the PC family(62, 75, 77). The same conclusions were also drawn for lung

cancer (11). Convertase overexpression can also alter the growth behavior and

the drug responsiveness in a human breast cancer cellline model (73).

In the past, 7B2 has been implicated with several types of neuroendocrine

tumors, such as neuroendocrine bronchial tumors, nonfunctioning pancreatic

islet tumors and ACTH-secreting pituitary tumors (94, 95), mainly

participating at the processing of tumor-secreted active peptides. Furthermore,

its concentration in the serum was associated with nonfunctioning pancreatic

islet cell tumors in the past (95). However, its role in tumorigenesis and

metastasis, especially in colorectal cancer, remains largely unknown.

Our data support the accumulation of active PC 1 and PC2 in metastasis

(Fig.10), and are in agreement with previous reports (3, 16). The main

question to date remains whether alterations ofPC1, PC2 and 7B2 expression

profiles are the cause or consequence of the metastatic phenotype.

It is weil known that PC1 and PC2 process pro-neurotensin to its active form,

neurotensin, which has been involved in colonic tumorigenesis (83, 112), or

pro-pancreatic peptide, proGHRH, proglucagon, prosomatostatin, and pro­

insulin to insulin (145, 149), which are known trophic factors for the gut. The

28 peptides and trophic factors are possibly involved in colonic tumorigenesis as weIl. Indeed, recently it was shown that PC 1 null mice are dwarfed, thus implicating PC 1 in the processing of GHRH and subsequent growth (145).

Therefore, change in PC 1 and PC2 expression and activation may alter the profiles of secretory proteins. This in tum could increase cell growth potential.

These changes could render the rest of the liver susceptible to future/further metastasis. Indeed, the fact that the PC2 profile observed in unaffected liver is partially different from the one observed in normal liver, supports the hypothesis that the tumor may induce modifications in the rest of the liver.

Whether PC 1, PC2 and 7B2 are directly implicated in such a model is under investigation.

In conclusion, the present study shows that PC 1, PC2 and 7B2 are present in human colorectal liver metastases, but also in unaffected and normal human liver tissue.

Furthermore, PC 1 and PC2 convertase expression and cleavage are altered in colorectal liver metastases, resulting in abundance of their biologically active isoforms. 7B2, whose overexpression in tumor is thought to play a key role in the above processes, at least for the processing of PC2, could represent a potential diagnostic, prognostic or even therapeutic target.

The role of ProSAAS is currently under intense investigation 10 our laboratory, since it has been implicated with the processing /c1eavage of the

PCI zymogen, but also due to its homology with 7B2 (55-59,150,151)

29 Acknowledgements

This study was performed under the guidelines of Mc Gill University Health

Center. The technical support by Shiquing Liu was very much appreciated.

The secretarial support of Mrs. Marie Monaghan cannot be overstated.

1 would like to thank Dr. Vicky Marcus that reviewed the

immunohistochemistry analysis.

1 am indebted to Dr. Nabil Seidah, for the generous gift of antibodies as weil

as for his scientific advise. 1 would also like to thank Dr. Chevet and Dr.

Jenna, for critically reviewing the manuscript and for their scientific help and

advise, without which this work would not be possible.

Finally, 1 would like to thank Dr. Peter Metrakos for his mentoring,

continuo us support and friendship during this endeavor.

30 Figure Legends

Figure l:The proprotein convertase family (PC).

Schematic representation of the family of the Proprotein Convertases (PCs),

indicating the tight phylogenetic conservation.

Figure 2: Expression of PCI, PC2 and GAPDH mRNA, in normal and

unaffected liver compared to colorectal (CRe) liver metastases.

A. PCR amplification products of PCI, PC2, and GAPDH, generated

after reverse-transcription in metastasis samples (T), their

corresponding unaffected liver samples (V) and normal liver samples

(N). Shown are the above products in seven tumor (TI-T7) and

unaffected (VI-V7) samples (out offourteen) and in three normal (NI­

N3) liver samples. Each experiment was done four times.

B. Quantification of PCI mRNA expression respectively, in 3 normal

(N), 14 tumor (T) and 14 unaffected (V) liver samples. Quantification

was obtained by the ratio of the optical density of PC 1 PCR

amplification products over GAPDH. Standard error of mean (SEM) is

indicated. Asterisk indicates a statistically significant difference

(P

31 C. Quantification of PC2 mRNA expression respectively, in 3 normal

(N), 14 tumor (T) and 14 unaffected (V) liver samples. Quantification

was obtained by the ratio of the optical density of PC2 PCR

amplification products over GAPDH. Standard error of mean (SEM) is

indicated. Double asterisk indicates a statistically significant difference

(P

Figure 3: Protein and processing/maturation profile o(PCl in normal,

una((ected and metastatic liver samples.

A. Schematic representation ofPCI structure. The antigenic region

against which the antibody was raised is mentioned (YAb). Cleavage

sites of the zymogen are indicated (arrows).

B. PCI cleavage profile in normal (NI-N3, top panel), unaffected (VI­

V2) and tumor (TI-T2, bottom panel) samples. Normalliver samples

(NI-N3) derived from three different organ donors. TIIUI and T2/V2

came from two independent patients. PCI immunoblotting was do ne

on a total of 3 normal, 14 metastasis and 14 unaffected liver samples,

and was repeated three times.

C. Relative amounts of total PCI (C, left panel) prote in, in tumor (T),

unaffected (V) and normalliver (N), expressed as a ratio over normal

liver samples. Ratios ofp84/p66 PCI isoforms (C, right panel) in the

32 same samples are calculated. SEM is indicated. Simple and double

asterisks indicate statistically significant differences (P<0.05).

D. E. Light microscopy PC 1 immunohistochemistry of liver metastasis

(T) and adjacent unaffected parenchyma (U), using 400 X

magnification. Arrowheads indicate positively stained cells in

unaffected liver. Empty arrow indicates positively stained tumor cells.

Figure 4: pe2 protein profile in normal. unaffected and tumor /iver samples.

A. Schematic representation of PC2 structure. The antigenic region

against which the antibody was raised is mentioned (YAb). Cleavage

sites are indicated (arrows).

B. PC2 c1eavage profile in normal (NI-N3, top panel), unaffected (UI­

U2) and tumor (Tl-T2, bottom panel) samples. Normal liver samples

(NI-N3) derived from three different organ donors. TIIUI and T2/U2

came from two independent patients. PC2 immunoblotting was do ne

on a total of 3 normal, 14 metastasis and 14 unaffected liver samples,

and was repeated three times.

Relative amounts of total PC2 (C, left panel) protein, in tumor (T),

unaffected (U) and normal liver (N)' expressed as a ratio over normal

liver samples. Ratios of p84/p66 PC2 isoforms (C, right panel) in

33 tumor (T), unaffected (U) and normal liver (N). SEM is indicated.

Double asterisk indicates statistically significant differences (P

Figure 5: Expression of 7B2 and GAPDH mRNA. in normal and unaffected liver compared to colorectal (CRe) liver metastases.

A. PCR amplification products of 7B2 and GAPDH, generated after

reverse-transcription in metastasis samples (T), their corresponding

unaffected liver samples (U) and normal liver samples (N). Shown are

the above products in seven tumor (Tl-T7) and unaffected (Ul-U7)

samples (out of fourteen) and in three normal (Nl-N3) liver samples.

Each experiment was done four times.

B. Quantification of 7B2 mRNA expression respectively, in 3 normal

(N), 14 tumor (T) and 14 unaffected (U) liver samples. Quantification

was obtained by the ratio of the optical density of 7B2 PCR

amplification products over GAPDH. Standard error of mean (SEM) is

indicated. Asterisk indicates a statistically significant difference

(P

34 Figure 6: 7B2 protein profile in normal, una((ected and tumor liver samples.

A. Schematic representation of 7B2 structure. The furin cleavage site is

shown (arrow). The antigenic region against which the antibody was

raised is indicated (Y Ab).

B. 7B2 immunoblots in normal liver (N1-N3, top panel), in tumor (Tl,

T2, bottom panel) and unaffected (U1, U2, bottom panel) liver.

Corresponding gels stained with Coomassie blue G250 (CS) are

shown. Experiments were repeated three times.

Figure 7: Light Microscopy immunohistochemistry of normal liver

stained with anti-PCI antibody.

A. Light microscopy of a section showing normal liver parenchyma,

stained with anti-PC1 antibody (see methods) using 100 X

magnification. Note the relatively low amount ofPCl. Arrows indicate

positively stained cells.

B. Light microscopy of the same section, stained with anti-PC 1 antibody

(see methods) using 400 X magnification. Arrows indicate positively

stained cells.

35 Figure 8: Light Microscopy immunohistochemistry of metastasis and unaffected Iiver stained with anti-PCl antibody.

A. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PCI antibody (see methods) using

25 X magnification. Note the relatively higher amount of PC 1 in areas

oftumor. Arrows indicate positively stained cens.

B. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PCI antibody (see methods) using

100 X magnification. Arrows indicate positively stained cens.

C. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PCI antibody (see methods) using

400 X magnification. Arrows indicate positively stained cens.

Figure 9: Light Microscopy immunohistochemistry of normal Iiver stained with anti-PC2 antibody.

A. Light microscopy of a section showing normal liver parenchyma,

stained with anti-PC2 antibody (see methods) using 100 X

magnification. Note the relatively high amount of PC2. Arrows

indicate positively stained cens.

36 B. Light microscopy of the same section, stained with anti-PC2 antibody

(see methods) using 400 X magnification. Arrows indicate positively

stained cells.

Figure 10: Light Microscopv immunohistochemistry of metastasis and unaffected liver stained with anti-PC2 antibody.

A. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PC2 antibody (see methods) using

25 X magnification. Arrows indicate positively stained cells. Note the

relatively equal amount of positively stained cells in tumor and

unaffected liver parenchyma.

B. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PC2 antibody (see methods) using

200 X magnification. Arrows indicate positively stained cells.

e. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-PC2 antibody (see methods) using

400 X magnification. Arrows indicate positively stained cells.

37 Figure 11: Light Microscopy immunohistochemistry of normal liver stained with anti-7B2 antibody.

A. Light microscopy of a section showing normal liver parenchyma,

stained with anti -7B2 antibody (see methods) using 100 X

magnification. Note the absence of 7B2 stained cells.

B. Light microscopy of the same section, stained with anti-7B2 antibody

(see methods) using 400 X magnification.

Figure 12: Light Microscopy immunohistochemistry of metastasis and

unaffected liver stained with anti-7B2 antibody.

A. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-7B2 antibody (see methods) using

25 X magnification. Arrows indicate positively stained cells. Note the

uniquely stained areas of tumor, compared to areas of unaffected liver

parenchyma, indicating a clear neuroendocrine pattern.

B. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-7B2 antibody (see methods) using

100 X magnification. Arrows indicate positively stained cells.

e. Light microscopy of a section showing tumor and adjacent unaffected

liver parenchyma, stained with anti-7B2 antibody (see methods) using

400 X magnification. Arrows indicate positively stained cells.

38 Figure 13: Schematic representation of the various amounts of pro­

PCI, PCI, pro-PC2, PC2 and 7B2 in tumor (T) and unaffected (U) samples.

Cartoon depicting the amounts of all the zymogens presented in the thesis, and their potential location within the cell; i.e. ER (endoplasmic

Reticulum), CGN (cis Golgi Network), Golgi apparatus, TGN (Trans

Golgi Network), SG (secretory Granules).

Figure 14:Schematic representation of the "neuroendocrine" subclone

hypothesis.

Cartoon showing the, above-mentioned neuroendocrine metastatic

subclone hypothesis. Specifically, from the primary tumor (to the left),

arises a subclone that possesses not only a metastatic potential but also a

neuroendocrine profile in terms of proprotein convertase 1 and 2. This

subclone is capable of passing through the extracellular matrix and

eventually give birth to liver metastases (to the right)

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63 EUBACTERIAE PLANTS 1 CMCUCU 1

YEAST

'?,.-\--'r~~:::::::::~-======hPACE4 F ~ hPC5

AcFurin

Ls-PC1 Ac-PC1

EUKARYOTIC PROPROTEIN CONVERTASES

Figure 1 A

PC1 PC2 GAPDH

Tumor Unaffected Normal

B PC1 0.7 r:::: .2 t/) 0.6 t/) Go) 1..c. 0.5 Go)>< oc:( 0.4 z 0:: 0.3 E """0 0.2 0 ;:; ca 0.1 0:: 0 T U N

C PC2 1.2 r:::: .2 t/) 1 t/) c.~ 0.8 >

T U N Figure 2 A t p84 ÀAb PC1 'é!.' Catalytic I>:::::~:>:::::~ 753 aa ..' ~ p66

B kDa

84 - 66 - lb: aPC1

T1 U1 T2 U2

84 - lb: aPC1 66 - C 4 12 3.5 * "r"" 10 ** 0 a. 3 (,Q (,Q 8 "'"0 2.5 Co s:: -:::::J 2 -~ 6 0 Co E cu 1.5 "'"0 0 4 CD ;:; ;:;> cu cu c::: 2 Qi 0.5 c::: 0 0 T U N T U N

Figure 3 A p75 ÀAb PC2 637 aa

B N1 N2 N3 75 - 66 - lb: aPC2

T1 U1 T2 U2

75 - lb: aPC2 66 - 1.2 1.6 c 1.4 N (J D.. 1.2 .... 0.8 Il')..... 0 c... c: (0 -:s 0.6 -(0 0.8 0 * ....c... E 0 0.6 cu 0.4 CI) 0 > +:i 0.4 +:i cu cu 0.2 c:: a:; 0.2 c:: 0 T U N T U N

Figure 4 A T1 T2 T3 T4 T5 TG T7

500 bp 782

500 bp GAPDH

U1 U2 U3 U4 US UG U7 500 bp ~ 782

500 bp GAPDH

N1 N2 N3

500 bp 782 500 bp GAPDH

B 2.5 c: .c;;0 t/) 2.0

eQ. >< Cl) 1.5 oC( Z 0::: E 1.0 """0 0 :;:: 0.5 cu 0::: 0.0 T U N

Figure 5 A ÀAb p21 782 186 aa +Furin B kDa N1 N2 N3

lb: a7B2

cs

T1 U1 T2 U2

lb: a7B2

cs

Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Active PC1 Active PC2

SG T U TGN :. NH, t' Golgi

QOOH CGN • i • NH, NH, ER U

Pro-PC1 Pro-PC2 782

Figure 13 Passage through "Altered " Extracellular matrix (neuroendocrine) PCs P ofile

1 "Classic" PCs Profile

Figure 14 APPENDIX

64 McGill University Health Centre

INFORMATION AND CONSENT FORM FOR STUDY PARTICIPANTS

Title: Functional Genomics and Proteomics of Colo-rectal Cancer and Hepatic, Pancreatic and Biliary Cancers: an integrative and comprehensive study. Principal Investigator: Dr. Peter Metrakos McGill University Health Center

BACKGROUND

The main objective ofthis research project is to understand how cancers of the intestine (colon and rectum), liver, pancreas and biliary system function on a genetic and protein level. Amongst the central challenges ofthese illnesses is the identification ofnew markers (e.g. like CEA and alpha-fetoprotein) to improve diagnosis and classification and to define new targets for more effective treatments. Another goal is to determine whether there are certain patterns of gene and protein expression that can predict the possibility of these cancers spreading ta other places in the body. At the current time, very little is known ofwhich genes are required for metastasis (spreading) to occur. This information may help in the clinical treatment of such illnesses in the future.

This research will involve the study of the following four different groups of patients whose current treatment, as assessed by their attending physician, involves having to undergo a biopsy and/or surgery: 1. patients diagnosed with colorectal cancer with or without liver metastases 2. patients diagnosed with hepatocel1ular carcinoma 3. patients diagnosed with neuroendocrine tumors with or without metastases 4. patients diagnosed with pancreatic cancer

STUDY PROCEDURE

Collection of Tissue

• During your biopsy and/or surgery, your doctor will remove sorne tissue from your body with which pathology tests will be performed. Your doctor will not take more tissue during surgery than needed for your care. • The results from these tests help assess your illness and will be used by your doctor to plan your subsequent care. Your doctor will give the results from these tests to you. • Once all the pathology tests have been performed, there may be sorne excess tissue left. Generally, this additional tissue is not kept because it will not affect your care, however it can be used to perform sorne of the research mentioned above. Vou may, therefore choose to have this tissue used for research purposes. The tissue will be subjected to genetic analysis to try to determine factors that cause disease, which may lead to new treatments in the future. • The tissue will be kept in frozen for up to five years, which is the length oftime this study will take to be completed. • Your tissue may be helpful for research whether you do or do not have cancer.

Version: 11126/2002 McGill University Health Centre

• There will be no direct health benefit to you from participating in this study. However, results from this work are likely to benefit people who have cancer and other diseases in the future.

B100d Collection

• In addition to using any left over tissue for analysis, we ask your consent to collect a small amount ofblood (20 mis or 4 teaspoons) from you while you are under anesthetic. • Three other samples of the same amount ofblood will be collected at the same time as blood is drawn at follow-up visits with your doctor. • There are several reasons why these samples are of great importance. A number ofblood tests are currently available to detect sorne types of cancer, such as the PSA test for prostate cancer. New tests are under development and it will be of great value to have a collection of blood specimens from patients to evaluate these tests. • We ask you to indicate your willingness to provide a blood sam pie below separately from your decision to allow us to use your tissue.

Will the decision to participate affect your care in any way?

• The choice to let us keep the excess tissue removed at surgery and/or to collect blood samples for future research is entirely up to you. No additional tissue will be taken and your care will not be affected by your decision.

Risks and benefits

• The tissue specimens taken for the purpose of research will be taken from those resected specimens that are in excess ofwhat is required for clinically relevant pathology tests. This is done in the Pathology suite; therefore such procurement represents no danger to you. • The tissue samples will be collected with the on-call pathologists present in the surgical pathology room. If there is any concern by the pathologist that diagnosis or the results of pathology tests will be compromised, tissue for purposes ofthis research will not be collected. • Blood collected for the purpose of analysis for new cancer markers will be carried out when blood tests are required for your continued treatment and follow-up purposes. Therefore, blood will not be drawn from you only for study purposes.

Confidentiality

• People that are trained to hand1e tissue and protect donor' s rights make sure that the highest standards of quality control are followed. • Samples collected during surgery are stored at the LD MacLean Laboratory. • Samples are provided only if the proposed research meet scientific and ethical standards. • The researchers will not be provided with your name, address, phone number, or any other information that may specifically identify you. • However, researchers may need to know the origins of the tissue samples in order to answer questions about diseases such as cancer.

Version: 11/26/2002 2 McGill University Health Centre

• The information that will be given to the researchers may include your age, sex, race, primary diagnosis, previous medical problems, and previous treatments. • This information will be collected from your medical records and will be sent to the researchers in a manner that do es not identify you. Your personal information is kept private and confidential by assigning a number to your tissue sample. • There will be no misuse of records. People not involved in the research will not have access to this information, which will further protect your privacy.

Compensation

• There is no financial compensation given to participants ofthis study at the current time.

Results from the study

• You will receive the results of your biopsy or pathology tests from your treating physician, but the results of the research done with your tissue are not likely to be available in the foreseeable future. Research can take a long time and involves the use of samples from a large number of people before conclusions can be drawn.

Voluntary Participation

• Participation in this study is completely voluntary. • You can change your mind about participating in this study at any time. If you decide you no longer want us to use your tissue for research, simply contact us. Your tissue will be discarded or destroyed. • Not participating will not compromise, in any way, the quality ofyour care.

Questions

Ifyou have any questions, please talk to your doctor, or cali the coordinator ofthis program Josie Campisi, at (514) 842-1231 ext. 36995. Ifyou have questions about your rights as a patient, contact the Royal Victoria Hospital ombudsperson, Pat Q'Rourke, at (514) 842-1231 ext. 35655.

Version: 11/26/2002 3 McGill University Health Centre

Please read each sentence below and think about your choice. Ifyou have any questions, please talk to Dr. Peter Metrakos at (514) 843-1600, or cali the coordinator ofthis program at the MUHC, Josie Campisi, at (514) 842-1231 ext. 36995.

Your Name and Contact Details:

Name (print)

Date of Birth

Address

Summarv

The following statements summarize the purpose of this research and what your participation entails. Read this section carefully. At the end, you can indicate whether you consent to these items.

1. Vou will receive a signed copy of the Information and Consent Form for Participants. The LD MacLean Laboratory will keep the original. 2. The investigators will conduct this study in a manner conforming to the ethical and scientific principles set out by the Canadian Institute ofHealth Research. 3. There will be no additional charges for your participation to this study, nor any financial benefit. 4. The LD MacLean Laboratory will retain excess tissue sampled during your biopsy or surgery. 5. The sample will be used for biological and genetic studies on the cause of cancer. 6. Participation in this study is completely voluntary. Retaining the excess tissue will not in any way compromise your care or the accuracy ofyour diagnosis. Vou may withdraw from the study at any time without prejudice, and if requested, your samples will be destroyed. Refusai to participate in this study will not affect in any way the treatment you will receive by your doctor or your nurse. 7. You may be asked to answer questionnaires. You may choose not to participate or answer any particular question. 8. Your medical records will be released in confidence to the investigators with the understanding that the records will be used only in connection with carrying out our obligations related to this study. Vou will not at any point be specifically identified. 9. Associated researchers will have access to the samples for approved research only. The researchers will not be able ta identify you or other family members. 10. Information about you will be entered in a Registry. Personal information will be treated with confidentiality, and will be identifiable only by an ID code. Il. The sample will be stored by the LD MacLean Laboratory. It will be stored in good faith, but its suitability for future research cannat be guaranteed. Samples will not be used for purposes other than those agreed to in this consent form.

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12. Twenty milliiiters or 4 teaspoons of peripherai blood will aiso be collected on four different occasions, separated and stored frozen at the LD MacLean Laboratory.

Doctor or Study Co-ordinator has explained the study to me and 1 understand the consequences involved in participating.

1 have read this Information and Consentform. YES NO

1 have had the opportunity to ask questions. YES NO

1 am satisfied with the answers to my questions. YES NO

1 consent to my participation and for my tissue to be used as described in sections 1-11 above. YES NO

1 consent to four small blood samples being collected from me. YES NO

Signature of participant:

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INFORMATION ET FORMULAIRE DE CONSENTEMENT DESTINÉ AUX PARTICIPANTS DE L'ÉTUDE

Titre: Génomique fonctionnelle et protéomique du cancer colorectal et des cancers hépatiques, pancréatiques et biliaire: une étude intégrative et exhaustive Investigateur: Dr Peter Metrakos Centre universitaire de santé McGill

HISTORIQUE

L'objectifprincipal de ce projet de recherche est de comprendre comment les cancers de l'intestin (colon et rectum), du foie, du pancréas et du système biliaire fonctionnent à un niveau génétique et protéique. Parmi les défis centraux de ces maladies, on retrouve l'identification de nouveaux marqueurs (par exemple l'antigène carcinoembryonnaire (ACE) et la alpha­ foetoprotéine (AFF)) dans le but d'améliorer le diagnostic et la classification et de définir de nouvelles cibles vers des traitements plus efficaces. Un autre objectif est de déterminer s'il existe des modèles d'expression de gène ou de protéine qui peuvent annoncer la possibilité de propagation des cancers vers d'autres endroits du corps. Présentement, nous connaissons très peu quels types de gènes sont requis pour que la métastase (propagation) se produise. Ces renseignements peuvent aider au traitement clinique de telles maladies à l'avenir.

Cette recherche comprendra l'étude des quatre groupes suivants de patients dont le traitement actuel, tel qu'évalué par leur médecin, nécessitera de subir une biopsie et/ou une chirurgie: 1. patients diagnostiqués du cancer colorectal avec ou sans métastase du foie 2. patients diagnostiqués d'un cancer primitif du foie 3. patients diagnostiqués d'une tumeur neuroendocrine avec ou sans métastases 4. patients diagnostiqués d'un cancer du pancréas

PROCÉDURE DE L'ÉTUDE

Collecte de tissu

• Pendant votre biopsie et/ou votre chirurgie, votre médecin prélèvera du tissu de votre corps et effectuera des analyses pathologiques. Votre médecin ne prendra que la quantité nécessaire de tissu pour vos soins pendant la chirurgie. • Les résultats de ces analyses aident à évaluer votre maladie et seront utilisés par votre médecin dans le but de planifier vos soins ultérieurs. Votre médecin vous donnera les résultats de ces analyses. • Une fois que toutes les analyses pathologiques seront effectuées, il pourrait y avoir des excès de tissus. En général, cet excès de tissu n'est pas conservé car il n'est pas utile à vos soins; toutefois, le tissu peut être utilisé dans le but d'effectuer certaines recherches mentionnées ci­ dessus. Vous pouvez alors choisir d'avoir le tissu utilisé à des fins de recherche. Le tissu sera sujet à des analyses génétiques avec l'objectif de déterminer les facteurs qui causent la maladie ce qui pourrait mener à de nouveaux traitements à l'avenir.

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• Le tissu sera maintenu congelé jusqu'à une période de cinq ans, ce qui représente la durée de toute cette étude du début à la fin. • Votre tissu pourrait aider la recherche que vous ayez le cancer ou pas. • Votre participation à cette étude n'apportera aucun bienfait direct à votre santé. Cependant, les résultats provenant de cette recherche sont susceptibles d'apporter des bienfaits aux personnes atteintes du cancer et d'autres maladies à l'avenir.

Prélèvement sanguin

• En plus d'utiliser de l'excès de tissu à des fins d'analyses, nous vous demandons votre consentement à prélever une petite quantité de sang (20 ml or 4 cuillerées à soupe) pendant que vous serez SQUS anesthésie. • Trois autres échantillons de la même quantité de sang seront prélevés en même temps que l'échantillon de sang lors des visites de suivi avec votre médecin. • L'importance considérable de ces échantillons comporte plusieurs raisons. Un nombre d'analyses sanguines est actuellement disponible afin de détecter certains types de cancer, tel que le cancer de la prostate avec l'analyse de l'antigène spécifique de la prostate. De nouveaux tests sont en voie de réalisation et ce sera d'une importance capitale d'avoir des échantillons de spécimens sanguins de patients dans le but d'examiner ces analyses. • Nous vous demandons de mentionner ci-dessous votre volonté de fournir un échantillon sanguin, une décision à part de celle qui nous permet d'utiliser votre tissu.

Est-ce que votre décision de participer affectera vos soins?

• Le choix de nous laisser garder le tissu excédentaire pendant la chirurgie et/ou de recueillir des échantillons de sang destinés à la recherche future dépend entièrement de vous. Aucun tissu additionnel ne sera prélevé et votre décision n'affectera pas vos soins.

Risques et bienfaits

• Les spécimens de tissus prélevés dans le but de la recherche seront pris à partir des spécimens réséqués excédentaires de ce qui est exigé pour les analyses pathologiques cliniquement pertinentes. Ceci est fait dans l'aire de pathologie; un tel prélèvement ne représente donc aucun danger pour vous. • Les échantillons de tissus seront recueillis devant la présence du pathologiste sur appel à la salle de chirurgie pathologique. Si le pathologiste s'inquiète que le diagnostic ou les résultats des analyses pathologiques risquent d'être compromis, il ne prélèvera pas de tissu dans le but de cette recherche. • Le sang prélevé dans le but d'analyser de nouveaux marqueurs de cancer sera effectué quand les analyses sanguines seront exigées pour votre traitement continu et avec l' obj ectif de suivi. Le sang qui vous sera prélevé ne sera donc pas uniquement destiné à l'étude.

Confidentialité

• . Les personnes formées dans le but de manipuler du tissu et de protéger les droits des donneurs s'assurent que les standards les plus élevés de contrôle de qualité sont suivis.

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• Les échantillons recueillis pendant la chirurgie sont entreposés au laboratoire LD MacLean. • Les échantillons sont fournis seulement si la recherche proposée est conforme aux standards scientifiques et éthiques. • Les chercheurs n'auront pas accès à votre nom, adresse, numéro de téléphone ou tout autre renseignement qui pourrait vous identifier spécifiquement. • Toutefois, les chercheurs pourraient avoir besoin de connaître les origines des échantillons de tissus dans le but de répondre aux questions sur des maladies tel que le cancer. • Les renseignements qui seront donnés aux chercheurs pourraient inclure votre âge, sexe, race, diagnostic primaire, antécédents de problèmes médicaux et traitements antérieurs. • Ces renseignements seront pris à partir de vos dossiers médicaux et envoyés aux chercheurs de manière à ne pas vous identifier. Vos renseignements personnels sont gardés privés et confidentiels en assignant un numéro à votre échantillon de tissu. • Vos dossiers ne seront pas employés abusivement. Les personnes qui ne participent pas à la recherche n'ont pas accès à ces renseignements ce qui protègera davantage votre vie privée.

Compensation

• Présentement, il n'y a aucune compensation financière donnée aux participants de cette étude.

Résultats provenant de l'étude

• Vous recevrez les résultats de votre biopsie ou analyses pathologiques de votre médecin traitant, mais les résultats de la recherche effectués avec votre tissu ne seront probablement pas disponibles dans un avenir prévisible. La recherche peut prendre beaucoup de temps et nécessite l'utilisation d'échantillons d'un nombre élevé de personnes avant d'en arriver à des conclusions.

Participation volontaire

• La participation à cette étude est entièrement volontaire. • Vous pouvez changer d'avis à propos de votre participation à cette étude à n'importe quel moment. Si vous décidez que vous ne voulez plus utiliser votre tissu pour la recherche, veuillez simplement communiquer avec nous. • Le fait de ne pas participer ne compromettra pas, d'aucune façon, la qualité de vos soins.

Questions

Si vous avez des questions, veuillez s'il vous plaît les poser à votre médecin ou appeler la coordonnatrice de ce programme, Josie Campisi, au (514) 842-1231 poste 36995. Si vous avez des questions à propos de vos droits en tant que patient, veuillez communiquer avec l'ombudsman de l'Hôpital Royal Victoria, Pat O'Rourke, au (514) 842-1231 poste 35655.

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Veuillez lire chaque phrase ci-dessous et réfléchir à votre choix. Si vous avez des questions, veuillez communiquer avec le Dr Peter Metrakos au (514) 843-1600, ou appeler la coordonnatrice de ce programme au CUSM, Josie Campisi, au (514) 842-1231 poste 36995.

Votre nom et coordonnées:

Nom (en caractères d'imprimerie)

Date de naissance

Adresse

Sommaire

Les énoncés suivants résument l'objectif de cette recherche et ce que votre participation nécessite. Veuillez lire attentivement cette section. À la fin, vous pourrez indiquer si vous consentez à ces énoncés.

1. Vous recevrez une copie signée du document intitulé Information et formulaire de consentement destiné aux participants. Le laboratoire LD MacLean gardera l'original. 2. Les investigateurs dirigeront cette étude en toute conformité aux principes d'éthiques et scientifiques entrepris par l'Institut de recherche en santé du Canada. 3. Votre participation à cette étude ne nécessitera aucun frais supplémentaire, ni de bénéfice financier. 4. Le laboratoire LD MacLean gardera du tissu excédentaire pris en échantillon pendant votre biopsie ou chirurgie. 5. L'échantillon ne sera pas utilisé à des fins d'études biologiques et génétiques sur le cancer. 6. Votre participation à cette étude est entièrement volontaire. Le fait de garder du tissu excédant ne compromettra d'aucune manière vos soins ou la précision de votre diagnostic. Vous pouvez vous retirer de l'étude à n'importe quel moment sans subir de préjudice, et si vous le demandez, vos échantillons seront détruits. Le refus de participer à cette étude n'affectera d'aucune façon le traitement que vous recevrez de votre médecin ou infirmière. 7. On pourrait vous demander de répondre à des questionnaires. Vous pouvez choisir de ne pas participer ou de ne pas répondre à une question en particulier. 8. Vos dossiers médicaux seront remis en toute confidentialité aux investigateurs et il est entendu que les dossiers seront seulement utilisés en relation avec la réalisation de nos obligations concernant cette étude. À aucun moment, vous ne serez identifié spécifiquement. 9. Les chercheurs associés auront accès aux échantillons seulement dans le but d'une recherche approuvée. Les chercheurs seront incapables de vous identifier ou d'identifier d'autres membres de votre famille.

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10. Vos renseignements seront inscrits à un registre. Vos renseignements personnels seront traités avec confidentialité et seront identifiables seulement par un code d'identité. Il. Les échantillons seront entreposés par le laboratoire LD MacLean. Les échantillons seront entreposés en bonne foi, mais on ne peut pas garantir qu'ils seront convenables à la recherche future. Les échantillons ne seront pas utilisés dans d'autres objectifs que ceux consentis dans ce formulaire de consentement. 12. Vingt millilitres ou 4 cuillerées à soupe de sang périphérique seront également recueillis à quatre occasions différentes, séparément et entreposés en congélation au laboratoire LD MacLean.

Médecin ou coordonnateur de l'étude m'a expliqué cette étude et je comprends les conséquences de ma participation.

J'ai lu ce document Information etformulaire de consentement OUI NON

J'ai eu l'occasion de poser des questions. OUI NON

Je suis satisfait des réponses à mes questions. OUI NON

J'accepte de participer et qu'on utilise mon tissu tel que décrit aux sections 1-11 ci-dessus. OUI NON

Je consens à ce qu'on me prélève 4 petits échantillons sanguins OUI NON

Signature du participant:

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