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Review |
From the Laboratory of Biochemical Neuroendocrinology,*Clinical Research Institute of Montreal, Montreal, Quebec; Molecular Medicine and Disease of Aging Center,Loeb Health Research Institute, Ottawa Civic Hospital, Ottawa, Ontario; and the Departments of Surgery and Medicine,McGill University and the Royal Victoria Hospital, Montreal, Quebec, Canada
Abstract
The mammalian subtilisin/kexin-like proprotein convertase (PC) family has been implicated in the activation of a wide spectrum of proteins. These proteins are usually synthesized as inactive precursors before their conversion to fully mature bioactive forms. A large majority of these active proteins such as matrix metalloproteases, growth factors, and adhesion molecules are crucial in the processes of cellular transformation, acquisition of the tumorigenic phenotype, and metastases formation. Inhibition of PCs significantly affects the malignant phenotype of various tumor cells. In addition to direct tumor cell proliferation and migration blockade, PC inhibitors can also be used to target tumor angiogenesis. In this Review article we discuss a number of recent findings on the clinical relevance of PCs in cancer patients, their implication in the regulation of multiple cellular functions that impact on the invasive/metastatic potential of cancer cells. Thus, PC inhibitors may constitute new promising agents for the treatment of multiple tumors and/or in adjuvant therapy to prevent recurrence.
, where n = 0, 2, 4 or 6 and X is usually not Cys.1-5
In contrast, the subtilisin kexin isozyme-1 (SKI-1) processes precursors at non-basic amino acids within the motif (R/K)-X-(L,V)-(L,T,K,F).1,6-9
Furin, PC5-B, PC7, and SKI-1 are the only members of the mammalian PCs with a transmembrane domain and cycle between the trans-Golgi network and the cell surface. These enzymes as well as PC5, PACE4, and PC4 are involved in the processing of proteins secreted via the constitutive pathway. In contrast, PC1 and PC2 are found within dense core secretory granules and process proteins secreted by the regulated secretory pathway.1-9
Like many other proteases, PCs are synthesized as inactive zymogens with an N-terminal prosegment extension. This conserved region is autocatalytically removed during PCs/SKI-1 maturation,1-7
by cleavage either at RXKR (for PCs) or for SKI-1 at the motif RX(V,L)(K,F,L). So far the only known substrates of the novel enzyme SKI-1/S1P6,7
are: probrain-derived neurotrophic factor,6
sterol regulatory element-binding proteins,7
the endoplasmic reticulum-stress response transcription factor ATF6,8
and the surface glycoprotein GP-C of Lassa virus.9
Thus, PCs are responsible for processing of neuropeptides, receptors, growth factors (GFs), cell surface glycoproteins, and enzymes, whereas SKI-1 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 bioactivity. The most common being the removal of carboxy-terminal basic residues by carboxypeptidase E or D (CPE, CPD).10 PCs in Cancers and Clinical Relevance
Multiple approaches, eg, 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.11-22
Although elevated expression of different PCs was reported for different human cancers and tumor cell lines,23-37
the relative importance of various PCs in these cancers has not yet been clarified. Tumor expression of PCs can be studied at the protein 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. Table 1
summarizes the results of studies on the expression of various PCs in human cancers and tumor cell lines. Early studies revealed a high furin expression in advanced lung tumors.23
Such an association has subsequently been confirmed in other malignancies such as breast,24
head, and neck25
cancers. Based on these studies furin expression in tumors may constitute a significant prognostic factor independent of other conventional clinicopathological ones. Other studies described a significant association between high expression of PC1 and PC2 in neuroendocrine tumors, suggesting their involvement in the malignancy of tumor cells with a neural and/or endocrine phenotype.26-28
Although these studies showed a positive association between PC1 or PC2 expression and the extent of tumors, further research is required to elucidate the importance and the prognostic role of these enzymes in endocrine-related cancers.
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PC5 and PC7 have been examined for their prognostic relevance in only a few human cancers (Table 1)
.24,29
These studies showed a positive association between PC7 expression and the extent of breast tumors of which PC5 was undetectable.24
In contrast, analysis of various human colon cancer cells, revealed the expression of PC5.29
The significance of these findings is at present not known.
In conclusion, what seems clear is that furin up-regulation is correlated with tumor progression and invasiveness. Further research is required to elucidate the prognostic role of the other PCs in various types of cancer.
PC Substrates in Tumor Growth and Metastasis
No report has yet appeared to indicate that the PCs could inactivate protein substrates or peptides in trans, even at high concentrations. The only exceptions are the inactivation of their inhibitory prosegment by an autocatalytic mechanism and the inactivation of the inhibitors pro-SAAS and 7B2 by their cognate enzymes PC1 and PC2, respectively. 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 critical proteins implicated in neoplasia, they may be targets of cancer therapy. Among these substrates, matrix metalloproteases (MMPs), GFs, and adhesion molecules through degradation of the extracellular matrix (ECM), modulation of cell growth, and/or migration are involved in tumor progression and metastasis (Table 2)
.
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The destruction of the basement membrane and ECM is associated with tumor cell invasion and metastasis. ECM is a complex structure that consists of collagen, proteoglycans, and other protein such as fibronectin, vitronectin, and laminin. Secreted proteinases from malignant and stromal cells degrade many ECM components, facilitating the detachment of these cells and their invasiveness. The level of expression of these proteinases in tumor cells is associated with advanced-stage tumorigenesis and poor prognosis. ECM degradation is a complex process involving a cascade of proteolytic events in which the primary step likely implicates enzyme activation by the PCs. The latter were reported to process the following metalloproteinases: stromelysin-3 (str-3),38,39 membrane-type MMPs (MT-MMPs),40 the adamalysin metalloproteinases (ADAMs),41 and the adamalysin metalloproteinases with thrombospondin motifs (ADAM-TS).42,43 The expression of these metalloproteinases has been correlated with increased local aggressiveness, metastasis, and poor clinical outcome.44-49
Stromelysin-3 (Str-3)
Str-3 is a member of the MMP family of which expression has been correlated with both increased local aggressiveness and poor clinical outcome.50
Activation of Str-3 by furin occurred intracellularly before secretion (Table 2)
.38,39
MT-MMPs
MT-MMPs, a new family of MMPs, are overexpressed in a wide variety of carcinomas, especially in the colon44,45
and brain51
and were reported to be involved in metastasis. Expression of these MMPs, particularly MT1-MMP was shown to process pro-MMP-2 thereby enhancing invasiveness both in vitro and in vivo.40,52
MMP-2 is the enzyme that degrades collagen IV, a major type of collagen in basement membranes. MT1-MMP possesses two typical recognition motifs for PCs, namely ArgArgProArg92
and ArgArgLysArg111
that were recently reported to be cleaved by furin-like enzymes at both sites (Table 2)
.40
Adamalysin Metalloproteinases
ADAMs are a family of membrane-associated multidomain zinc-dependent metalloproteinases with high sequence homology and domain organization, similar to the snake venom metalloproteases of the adamalysin subfamily.46
Sixteen of the 30 ADAM proteins identified to date are predicted to be catalytically active, based on the presence of a conserved zinc-binding sequence (HEXXH) in the protease domain, whereas the other members are not likely to be active proteases. The prodomains of several ADAM proteins such as ADAM12 are constitutively cleaved by a furin-type PCs as they progress through the secretory pathway (Table 2)
.41
This family was reported to play a role in diverse biological processes such as fertilization, myogenesis, neurogenesis, and cell surface proteolysis and shedding of different proteins.41,47,53-59
Of the proteins shed by the ADAMs, are cytokines and GFs such as transforming growth factor-
(TGF-), epidermal growth factor (EGF), heparin-binding EGF, tumor necrosis factor- (TNF-), c-Kit-ligand-1 (KL-1), colony-stimulating factor-1 (CSF-1), and Fas-ligand (Fas-L), receptors such as TNF receptor-I (TNFR1, p60 TNFR), TNF receptor-II (TNFR2, p80 TNFR), p75 nerve growth factor receptor (p75NGFR), interleukin-6 receptor (IL-6R), thyroid-stimulating hormone receptor, adhesion molecules such as L-selectin, and others proteins such as protein tyrosine phosphatase 
(PTP), protein tyrosine phosphatase LAR (LAR), amyloid precursor protein, and angiotensin-converting enzyme.53,56
Certain released molecules can be cleaved by more than one enzyme, and some enzymes can cleave more than one substrate. For example, cleavage of TNF- can be mediated by ADAM17 (TACE) and ADAM10, and -secretase activity for amyloid precursor protein has been attributed also to ADAM17,54
ADAM9,57
and ADAM10.59
It is not known how these protease(s) select their substrate because reliable consensus cleavage sites have not been identified. In addition to their proteolytic function, some members of the ADAM family such as ADAM15 and ADAM2 can support integrin binding via their disintegrin domain.58,60,61
It is increasingly recognized that ADAMs represent a novel group of membrane proteases that are important for cellular interactions under physiological and pathophysiological conditions including cancer. Recently, several ADAM family members were described to be dramatically up-regulated in many tumor cells. This includes cells derived from a range of hematological malignancies47
and breast, prostate, lung, and colon cancer.42,48,62
Therefore, it is of particular interest that the member of the ADAM family reported to shed cell-associated neural adhesion molecules such as L1 may be relevant to promote cell migration and invasion.62,63
Interestingly, in these cells the putative tumor suppressor gene MDC (ADAM11) was expressed at a very low level.62,63
Adamalysin Metalloproteinases with Thrombospondin Motifs
ADAM-TSs are a new member of the ADAM family containing thrombospondin-type motifs. They consist of multiple domains of proteins common to the ADAM family, including pro-, metalloprotease-like, and disintegrin-like. The first member of this family, called ADAM-TS1, was originally cloned from a colon adenocarcinoma cell line.42
Based on its capacity to form a covalent complex with 2-macroglobulin, recent studies demonstrated that ADAM-TS1 protein is proteolytically active (Table 2)
.43
In addition, the maturation of ADAM-TS1 precursor is impaired in the furin-deficient cell line, LoVo, and the processing ability of the cells is restored by the co-expression of the furin cDNA.64
The only members of the ADAM-TS family with established substrates are ADAM-TS2 (procollagen-N-proteinase) and ADAM-TS4 and -TS11 (aggrecanases-1 and -2, respectively).42,64,65
These proteinases cleave aggrecan at one or more of five specific sites in the aggrecan core protein.42,64,65
Recently, brain-enriched hyaluronan binding (BEHAB)/brevican, a brain-specific ECM protein was reported to be processed by ADAM-TS4. The processed form of BEHAB/brevican is dramatically increased in human gliomas, a notoriously invasive tumor.49
The rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms noninvasive tumors when grown as intracranial grafts, can form invasive tumors when it is transfected with a 5' cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA.49
Interestingly, the expression of ADAM-TS4 is induced during endothelial cells undergoing differentiation into tube-like structures suggesting its implication in angiogenesis.66
These observations link ADAM-TS family members to invasion and the blocking the activation of ADAM-TSs by PC inhibitors may provide a novel therapeutic strategy.
Adhesion Molecules
Cell adhesion molecules (CAMs) are cell-surface proteins that mediate cell-cell and/or cell-ECM interactions. They control cellular traffic, transmigration through the endothelium, homing in and localization to various target organs during inflammation, and tumor cell colonization.67,68 Most of the CAMs characterized so far fall into three categories of proteins: the immunoglobulin (Ig), integrin, or selectin families.
The Immunoglobulin Family
The Ig family includes intercellular adhesion molecules ICAM-1, ICAM-2, and ICAM-3, vascular CAM-1 (VCAM-1), and mucosal addressin CAM-1 (MadCAM-1), none of which are believed to be substrates for PC processing. However, the convertases seem to be required for their expression and probably function. Indeed, the expression of ICAM-1 and VCAM-1 on endothelial cells is induced by various cytokines and GFs such as interferon-
, interleukin-1 (IL-1), TNF-, insulin-like growth factor (IGF-1), and endothelins.69-76
Some of these precursors were reported to be directly processed by PCs such as IGF-1 and endothelins (Table 2)
.77-79
Others are indirectly regulated by the PCs through the cleavage of their cognate enzymes, for example TNF- converting enzyme (TACE)80
and ADAM1056
that process pro-TNF- are themselves activated by PCs.59,80,81
The Integrin Family
Its now well established that integrins are implicated in tumor progression and metastasis.82,83
Integrins are heterodimeric ß transmembrane receptors that bind ligands such as the ICAMs, VCAMs, and several ECM components. The extracellular domains of both subunits are required for ligand binding whereas the cytoplasmic tails interact with the cytoskeleton, induce changes in cell shape and motility, and transduce growth and survival signals.84,85
In addition, activation of integrins was reported to mediate MMPs and urokinase activity of many tumor cells, including melanoma and colon carcinoma.86-88
Although ß-subunits are not cleaved by PCs, a total of 9 of 18 known -subunits possess a potential PC-cleavage site, with 3, 4, 5, 6, and v proven to be PC5 and furin substrates (Table 2)
.89,90
Recently, Berthet and colleagues91
reported that the endoproteolytic cleavage of v integrin is important for the signal transduction pathway mediating cell adhesion. Blockade of vß5 cleavage resulted in a decreased phosphorylation of focal adhesion kinase (FAK) and paxillin, two important molecules involved in cell adhesion.
The Selectin Family
The selectin family members, L-selectin, P-selectin, and E-selectin are involved in the adhesion of leukocytes to activate the endothelium. Adhesion is initiated by weak interactions that produce a characteristic rolling motion of leukocytes on the endothelial surface. This rapid on-off attachment is necessary for activation and engagement of integrins and their counterreceptors on the leukocytes and endothelial cells, respectively. The integrin-mediated, high-affinity binding is in turn required for leukocyte arrest and extravasation.67
On the basis of in vitro studies it is postulated that similar cell-cell interactions may also occur between circulating malignant cells and the vascular endothelium during tumor dissemination.92-94
In general, the selectins bind sialylated, glycosylated, or sulfated glycans on glycoproteins, glycolipids, or proteoglycans.95
The tetrasaccharides sialyl-Lewisx (sLewx) and sialyl-Lewisa (sLewa) are recognized by all three selectins. In vitro adhesion studies showed that human colorectal, pancreatic, and gastric carcinoma cells use sLewx and related carbohydrate determinants to adhere to TNF--inducible E-selectin on cultured vascular endothelial cells.93,96-98
In vivo studies in turn showed that inhibition of liver metastasis of the highly metastatic human pancreas adenocarcinoma (PCI) cells could be blocked with antibodies to sLewa and that lung colonization by colon carcinoma HT29 cells could be blocked by a soluble E-selectin fusion protein.97,98
Under normal physiological conditions, vascular endothelial cells express low constitutive levels of E-selectin. Several cytokines and GFs such as IL-1, TNF-, vascular endothelial growth factor, IGF-1, and endothelins induce the expression of E-selectin.75,77,99,100
Many of these molecules directly or indirectly require PC activity (Table 2)
. Thus, although like VCAMs and ICAMs, selectins are not processed by PCs, the latter may indirectly control their expression via the activation of some of the above inducers. Recently, we demonstrated that E-selectin expression on sinusoids was described to be one of the early molecular events involved in metastasis.101
The arrest of tumor cells in the hepatic circulation causes a cascade of events, which start with a rapid release of IL-1 and TNF- (and other mediators). In turn, these stimulate the expression of E-selectin on hepatic endothelial cells, resulting in an enhanced tumor cell adhesion and subsequent metastases.101
GFs and Their Receptors (GFRs)
The availability of GFs is critical for malignant transformation and metastasis. These molecules mediate cell entry into, and progression through, the cell cycle. They are divided into two main categories namely, competence factors such as platelet-derived growth factor, vascular endothelial growth factor, and basic fibroblast growth factor that enable cells to enter the G1 phase and progression factors such as IGF-1 that are required for progression from G1 into S phase and ultimately resulting in cell division.102-105
Many of these proteins as synthesized as proproteins that are processed and activated by PC-like enzymes (Table 2)
. Parallel increased expression of both GFs and PCs may result in tumor growth. Of the GFs shown to be processed by PCs include IGF-1, endothelins, nerve growth factor, PTH, and TGF-ß (Table 2)
.77,78,106-108
Others, are suspected to be PC substrates based on the presence of the RXXR motif, eg, FGF23 (Table 2)
109
that is reported to be involved in tumor-induced osteomalacia. Many GFs mediate their effects through receptor tyrosine kinases (RTKs) that transmit signals to the nucleus through an intricate network of adapter and signaling molecules.104
Receptor activation through ligand binding generally induces receptor dimerization and autophosphorylation through a trans mechanism.104,110
Transphosphorylation at specific tyrosine residues in turn generates binding sites that are recognized by proteins involved in cellular signaling. Several proteins that are phosphorylated through GF-associated kinase activity were identified as downstream mediators of receptor-associated signal transduction include: phospholipase Grb,111
C (PLC),112
GTPase-activating protein,113
phosphatidylinositol 3-kinase (PI-3 k),114
insulin receptor substrate (IRS), and homology and collagen (Src).115,116
RTKs are implicated in malignant transformation and tumor progression.117,118
Overexpression or mutations/insertions in GF receptors (GFRs) that result in constitutively high levels of proteins, or active kinases are documented in many tumors.119,120
Frequently this is accompanied by constitutive high expression of the respective ligands providing an autocrine mechanism for growth autonomy.121
Several GFRs contain a consensus PC-cleavage site, and are critical for tumor growth and metastases, eg, the HGF receptor (HGF-R),122
insulin receptor,123-125
and IGF-1 receptor.20,126
Hwang and colleagues,125
reported that cleavage of insulin receptor is essential in the signal transduction of insulin. In agreement with this observation, we recently showed that the uncleaved IGF-1 receptor is unable to undergo the critical steps for IGF-1-mediated growth and survival of tumor cells, ie, IGF-1-sensitive autophosphorylation and IRS-1 phosphorylation.20
However, Komada and colleagues122
previously reported that both cleaved and uncleaved HGF receptors can bind HGF and mediate their autophosphorylation and cell growth. This unusual result indicates that proteolytic processing of this receptor may not be essential for some of the tested signal transduction pathways of HGF. However, to better define the role of PC-mediated cleavage of this receptor, more extensive analysis is required.
PC Inhibitors
The potential clinical and pharmacological role of the convertases has fostered the development of both peptide- and protein-based PC inhibitors.1-5
The most promising protein-based specific inhibitors of PCs are a 1-anti-trypsin variant known as 1-anti-trypsin Portland or 1-PDX127,128
and the individual PC prosegment-based inhibitors.129,130
The 1-Anti-Trypsin Variant, 1-Anti-Trypsin Portland (1-PDX)
Like furin substrates, inhibitors of furin also require the interaction of enzyme subsites with the basic residues of the substrate. Previously, the trypsin inhibitor and the third domain of turkey ovomucoid have been reported to be inhibitors for furin.131,132
However, their equilibrium constant was representative of a moderate, rather than a potent, inhibitor. Inhibition of furin in the subnanomolar range was accomplished by bioengineering the 1-anti-trypsin variant, 1-PDX (1-anti-trypsin Portland).127
Kinetic analysis shows that a portion of bound 1-PDX operates as a suicide inhibitor.128
Once bound to furin’s active site, 1-PDX can either undergo proteolysis by furin or form a kinetically trapped sodium dodecyl sulfate-stable complex with the enzyme.128,133
In an attempt to produce a specific furin inhibitor others researcher mutated the bait region of the general protease inhibitor 2-macroglobulin (RVGFYESDVM690 into RVRSKRSLVM690).134
This variant was reported to inhibit processing of furin substrates, eg, human immunodeficiency virus type-1 glycoprotein gp160, von Willebrand factor, and TGF-ß1 was inhibited in COS-1 cells.134
The ovalbumin-type serpin human proteinase inhibitor-8, containing two instances of the minimal furin recognition sequence (VVRNSRCSRM343), has also been shown to inhibit furin in a rapid, tight binding manner that is characteristic of physiological serpin-proteinase interactions.135
However, the cytosolic localization of inhibition inhibitor-8 requires the addition of a signal peptide before it could inhibit furin in vivo, and even then its ex vivo inhibitory property is yet to be proven. More recently Cameron and colleagues136
reported that although the hexa-D-arginine is a potent and relatively specific furin inhibitor, the reduced ability of this highly charged peptide to cross the cell membrane for in vivo therapeutics is problematic. Based on these studies, to date the most promising protein-based specific inhibitor of PCs is the 1-anti-trypsin variant, 1-PDX. This serpin was first showed to be a potent inhibitor of furin-mediated cleavage of HIV gp160,127,137
but subsequently demonstrated to also inhibit all PCs involved in processing within the constitutive secretory pathway.138-140
Recent studies showed that endogenous inhibition of precursor convertases by 1-PDX reduces the production of the amyloid precursor -secretase product amyloid precursor protein-59-141
and blocks the activation of the pore-forming toxin proaerolysin,142
the maturation of the surface glycoproteins of infectious pathogens,127,128,137
the proteolytic activation of bone morphogenetic protein-4 (BMP-4),143
the cleavage of Notch,144
insulin-like growth factor-1 receptor (IGF-1R),20
and TGF-ß.145,146
The potential therapeutic value of PC inhibitors was recently reinforced by a report showing that exogenous addition of 1-PDX potentially inhibits the furin-dependent processing of human cytomegalovirus envelope glycoprotein gB, thus reducing the titer of infectious hCMV more effectively than currently used anti-herpetic agents.128
As uptake of 1-PDX into the cell could not be detected in cell lines lacking the enzyme, it was suggested that the reported furin inhibition by the external application of 1-PDX occurs because furin is localized to the trans-Golgi network and cycles to the cell surface, where it could meet 1-PDX, and back via endosomal compartments.128
A similar mechanism was attributed for the prevention of Pseudomonas exotoxin A activation when extracellular 1-PDX was applied to A7 melanoma cells.128
These studies collectively suggest that inhibition of PCs localized in the interior of the cell can be initiated at the extracellular cell surface by PC binding to 1-PDX and uptake, resulting in inhibition of pathological disease processes including cancer.
1-PDX and Tumor Cell Malignant Phenotypes
The in vitro and in vivo studies recently reported by our group and others demonstrated that the serpin 1-PDX could be a powerful tool for tumorigenesis inhibition.21,20
In ex vivo studies, using human leukemia Jurkat T and colon carcinoma HT29 cell lines and mouse pituitary tumor AtT20 cells, we found that stable expression of 1-PDX provoked dramatic changes in several malignant phenotypes and metastatic potential. This includes cellular growth, survival, invasiveness, and tumorigenesis.
The Role of PCs in Cell Growth
The availability of GFs is critical for malignant transformation and metastasis. The anti-proliferate effect of 1-PDX on tumor cells is probably because of the inhibition of the processing of various proteins, including GFs and/or their corresponding receptors.77,147-150
Previously, it was postulated that the critical nature of the furin processing of various precursors may explain the anti-proliferative effect of furin blockade on H-500 rat Leydig tumor cells,12
the pancreatic ß-cell line MIN6,16
and the gastric mucus cells.15
Kayo and colleagues16
showed that conditioned medium derived from furin-overexpressing MIN6 cells stimulated the growth of their parental control cells, whereas the medium from cells expressing 1-PDX resulted in a lower growth rate. These results suggest that high furin expression stimulates growth through an autocrine/paracrine mechanism. In agreement with this hypothesis our group recently demonstrated the importance of the processing of IGF-1 receptor in the mediation of IGF-1-mediated tumor cell proliferation.20
A functional IGF-1R is known to be required for cell growth of various transformed cells. Overexpression and/or constitutive activation of IGF-1R in a variety of cell types leads to ligand-dependent growth in serum-free medium and to the establishment of a transformed phenotype such as the ability to form colonies in soft agar and tumors in mice.118,151
Similarly, studies reported on TGF-ß1 processing by PCs revealed that inhibition of TGF-ß1 in glioblastoma and in head neck squamous cell carcinoma (HNSCC) cell lines by 1-PDX could be a promising tool in modulating tumor growth and immunotherapy.21,22
The Role of PCs in Cell Survival
Similarly to PCs role in growth, PC cell-survival function is also substantial. Thus, the expression of 1-PDX in tumor cells exaggerates the apoptotic phenotype (DNA degradation and propidium iodide-positive cells) after serum deprivation, as we reported for HT-29 and Jurkat leukemia cells.20
The role of PC in cellular survival can be explained by their role in the maturation of various proteins known to be anti-apoptotic mediators participating in various autocrine/paracrine mechanisms. A likely scenario may involve one or more secreted ligands and/or their receptors, all of which require processing by PC-like enzymes. Examples include IGF-1 and IGF-2 that are synthesized and secreted by these cells152
and processed by furin.148,149
Because overexpression of 1-PDX in tumor cells inhibited the processing of IGF-1R and its furin-processed ligands, IGF-1 and IGF-2, it is liable to abrogate their autocrine/paracrine protective effects. The protective effect of many proteins is apparently dependent on their ability to induce a cascade of events leading to downstream effector pathway phosphorylation, including FAK,153,154
PI-3K1,155
and IRS-1.156
After phosphorylation these molecules mediated their anti-apoptotic effect through the activation of several negative death regulators such as Bcl-2 or inhibition of IL-converting enzyme-like caspases.153-157
Interestingly, expression of 1-PDX in tumor cells resulted in a reduction of basal tyrosine phosphorylation of FAK, PI-3K, and IRS-1.20
Exogenous addition of FAK, PI-3K, and IRS-1 activators such as IGF-1, failed to increase tyrosine phosphorylation of these molecules in tumor cells and to rescue the cells from apoptosis. This suggests a blockade in the transmission of the autocrine/paracrine anti-apoptotic signals in these cells.
The Role of PCs in Cell Invasion
PCs role in in vitro invasion is now well documented. Inhibition of PCs in different tumor cells resulted in a significant reduction in their invasiveness.20,25
This reduction is because of processing blockade of proteins directly involved in the mechanism of invasion such as MMPs or to proteins such as GFs and/or integrins reported to induce the expression of ECM-degrading protein including urokinase and MMPs.20,25,38,39,40-43
Recently, the inhibitory effect of 1-PDX on in vivo invasion was reported by Bassi and colleagues.21
They used an in vivo invasion assay based on the penetration of tumor cells into the tracheal wall. When transplanted in the tracheas the control-transfected cells penetrated deeply into the tracheal wall and reached the adventitia and the surrounding peritracheal tissues. Whereas, the penetration of 1-PDX-transfected cells in the tracheal wall was markedly decreased and never reached the outer surface of the trachea. They estimated this in vivo invasion reduction to be 
70 to 80%. We also found that 1-PDX decreased the invasiveness of colon carcinoma HT-2920
in which the processing of MT1-MMP was inhibited (Figure 1)
. In these cells the mRNA level of plasminogen activator urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), the urokinase-type plasminogen activator receptor (uPAR), and the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1), molecules involved in invasion processes and believed not to be processed by PCs, was significantly reduced. This data demonstrated both the direct and the indirect involvement of PCs in tumor cell invasion.
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The anti-tumor effects of PC inhibition was initially reported by our group. Expression of 1-PDX in colon carcinoma HT-29 tumor cells delayed the appearance, the incidence, and the vascularization of palpable tumors.20
Subsequently, the study reported by Bassi and colleagues21
have confirmed the inhibition of tumor growth by PC inhibition and found that the levels of furin mRNA and protein expression correlate with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. These studies demonstrated that the inhibitory effect of 1-PDX on tumor growth and invasiveness in in vivo systems may be underestimated because of the significant loss in 1-PDX expression in the subcutaneous tumors during their growth. However, it is not clear whether the observed progressively lower levels of 1-PDX mRNA in tumors21
is because of a specific loss of the cDNA from tumor cells. Future studies should address this issue by comparing the levels of 1-PDX to those of a specific marker co-expressed with this serpin, eg, green fluorescent protein. This may eliminate the trivial explanation of a dilution effect of the original tumor cells by host cells. However, similar observations were reported by Leitlein and colleagues,22
on the loss of 1-PDX expression ex vivo. These authors claim that 1-PDX expression was not very stable in glioma cell lines and was lost within a few passages in vitro. Angiogenesis is a significant prognostic factor in various cancers, but the factors that control angiogenesis in vivo are not well defined. Usually tumor angiogenesis is mediated by tumor-secreted angiogenic GFs that interact with their surface receptors expressed on endothelial cells. Multiple angiogenic proteins are known, including vascular endothelial growth factor and its four isoforms (121, 165, 189, and 206 amino acids), TGF-ß1, pleiotrophin, acidic and basic fibroblast growth factor. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells revealed a reduced tumor vascularization of tumors developed from tumor cells expressing 1-PDX (Figure 2)
.20
This suggests the importance of the PCs in tumor vessel formation through direct/indirect activation of various angiogenic proteins.
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To date the only naturally occurring intracellular PC inhibitor found in the constitutive secretory pathway are PC’s own propeptides or prosegments,2,6,158
and in the case of PC1 its C-terminal domain.159
The activities of the regulated secretory pathway convertases, PC1 and PC2, are however regulated by selective and specific inhibitors known as pro-SAAS160,161
and 7B2,162
respectively. For many proteins, the prosegment serves as an intramolecular chaperone that is essential for their correct folding,163
or/and transport and secretion.164
In the case of many proteolytic enzymes such as cathepsins, carboxypeptidases, papain, and subtilases, this prosegment was reported to be a very potent inhibitor that is highly specific for its associated protease. In these enzyme systems the prosegment is still an effective inhibitor even after initial proteolysis and in most cases an additional cleavage within the prosegment to release it and thereby to fully activate the enzyme.158,165
In accordance with the notion that PCs are initially produced as zymogens and proteolytically activated, Anderson and colleagues129
demonstrated that the prosegment of furin when used as a fusion protein to glutathione S-transferase exhibits a potent in vitro inhibitory activity on furin.165
We have also shown that the prosegment of furin and PC7 purified from bacterial culture media, are very potent inhibitors of their respective enzymes. Additionally, in vitro studies demonstrated the synthetic peptides derived from the prosegments of PC1, PC7, and furin are potent inhibitors of their enzymes.129,130,166,167
Based on these data, we surmised that, aside from 1-PDX, the PC prosegments may be applied in cancer therapy, as they were in Alzheimer’s disease ß-secretase analysis.168
Indeed these prosegments were shown to inhibit intracellularly the processing of GF precursors such as nerve growth factor and brain-derived neurotrophic factor,129
both of which have been linked to neuronal cancer and metastasis.169
In addition, we recently demonstrated that the cellular processing of IGF-1R, known to be crucial for the malignant phenotype of tumor cells,20,118,151
is effectively blocked by 1-PDX20
as well as by the prosegments of furin and PC5 but not by that of PC7 (Figure 3)
. Although some data exist to show that 1-PDX does not significantly inhibit thrombin,127
these are not exhaustive and the ability of 1-PDX to inhibit other classes of proteases is yet to be fully elucidated. In addition, because prosegment inhibitors are not always completely selective for their cognate enzymes and can inhibit other PCs,129
it will be necessary to modify their structure to improve their selectivity without compromising their potency (N. Nour and N. G. Seidah, in preparation).
|
Regulation of cellular proliferation, differentiation, and adhesion are complex processes in which different biological systems interact. Disruption of these processes is a hallmark of malignant transformation resulting in tumor progression and acquisition of a metastatic potential. The key to successfully developing efficient clinical strategies against cancers is to target a protein known to be essential in the growth of tumors and to validate that the disruption of this target selectively blocks the growth of the transformed cells. In this Review we have described recent progress made in establishing a novel approach to inhibiting tumor growth and tumor cell malignant phenotypes. We have outlined PC inhibition as a strategy to simultaneously disrupt the function of numerous proteins involved in the acquisition of the invasive/metastatic potential of cancer cells (Figure 4)
. The potential of this approach has been firmly established by the use of PC inhibitors (1-PDX and PC-prosegments) targeting the activity of the PCs. The cumulative data available established that 1-PDX is not only able to block the interaction of GFs and their receptors such as IGF-1/IGF-1R, and the activation of several MMPs but also to inhibit tumor cell proliferation and invasion (Figure 4)
. This potent effect further translates into an ability to slow down the growth of human tumors in a nude mouse model through an anti-proliferative and anti-angiogenic mechanism (Figure 2)
.20,21
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Acknowledgements
We thank Mrs. Edwige Marcinkiewicz for her assistance during the immunohistochemistry analysis.
Footnotes
Address reprint requests to Abdel-Majid Khatib, Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec, Canada H2W 1R7. E-mail: khatiba{at}ircm.qc.ca
Accepted for publication March 13, 2002.
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