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.

Proproteins are the fundamental units from which bioactive proteins and peptides are derived by limited proteolysis. Precursors are usually cleaved at the general motif (K/R)X n (K/ R)2, where n ϭ 0, 2, 4, or 6 and X is usually not a Cys. Seven dibasic specific mammalian proprotein convertases (PCs) 1 have been identified: furin, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, and PC7/LPC/PC8. Each of these enzymes, either alone or in combination with others, is responsible for the tissue-specific processing of multiple polypeptide precursors. This combinatorial mechanism generates a large diversity of bioactive molecules in an exquisitely regulated manner (1-4). Some of these precursors include adhesion molecules, e.g. integrin ␣-subunits (5); matrix metalloproteinases (MMPs) such as stromelysin-3 and membrane-type MMPs (MT-MMPs) (6, 7); several growth factor precursors, including transforming growth factor-␤ (8, 9), insulin-like growth factor-1 (IGF-1), and IGF-2 (10 -12); and some growth factor proreceptors such as the insulin receptor (13) and phosphotyrosine phosphatase (14).Recently, the potential clinical and pharmacological role of the convertases fostered the development of both peptide-and protein-based PC inhibitors (for reviews, see Refs. 1-4). The most promising protein-based specific inhibitors of PCs is an ␣ 1 -antitrypsin variant known as ␣ 1 -antitrypsin Portland (␣ 1 -PDX) (15-17) and the individual PC prosegment-based inhibitors (18). Recent studies showed that inhibition of PCs by ␣ 1 -PDX reduces the production level of the amyloid precursor ␣-secretase product ␤-amyloid precursor protein-␣ (19) and blocks the activation of the pore-forming toxin proaerolysin (20), the cleavage of Notch (21), the proteolytic activation of bone morphogenic factor-4 (22), and the maturation of the surface glycoproteins of infectious viruses (15,17,23 1 The abbreviations used are: PCs, proprotein convertases; MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; IGF, insulin-like growth factor; IGF-1R, insulin-like growth factor-1 receptor; ␣ 1 -PDX, ␣ 1 -antitrypsin Portland; uPA, urokinasetype plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; IRS-1, insulin-related substrate-1; FCS, fetal calf serum; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; bp, base pair; SKI-1, subtilisin kexin isozyme-1.

The physiological role of the subtilisin/kexin-like proprotein convertases (PCs) in rodents has been examined through the use of knockout mice. This review will summarize the major in vivo defects that result from the disruption of the expression of their genes. This includes abnormal embryonic development, hormonal disorder, infertility, and/or modified lipid/sterol metabolism. Members of the PC family play a central role in the processing of various protein precursors ranging from hormones and growth factors to bacterial toxins and viral glycoproteins. Proteolysis occurring at basic residues is mediated by the basic amino acid-specific proprotein convertases, namely: PC1/3, PC2, furin, PACE4, PC4, PC5/6, and PC7. In contrast, proteolysis at nonbasic residues is performed by the subtilisin/kexin-like isozyme-1 (SKI-1/S1P) and the newly identified neural apoptosis-regulated convertase-1 (PCSK9/NARC-1). In addition to their requirement for many physiological processes, these enzymes are also involved in various pathologies such as cancer, obesity, diabetes, lipid disorders, infectious diseases, atherosclerosis and neurodegenerative diseases.

The liver is a major site of metastases for some of the most common human malignancies, carcinomas of the gastrointestinal tract in particular. Liver metastases are frequently inoperable and are associated with poor prognosis. 1 The metastatic cascade involves a sequence of steps that can lead to tumor cell arrest in the vascular bed of an invaded organ such as the liver and subsequently to tumor extravasation into the extravascular space.2 These events are regulated by, and in turn, can induce host proinflammatory responses that involve tumor-and host-derived chemokines and cytokines. A key mediator of the inflammatory response is tumor necrosis factor (TNF)-␣. This cytokine can play a dual role in tumor progression and metastasis. On one hand it can inhibit tumor growth through its cytocidal and proapoptotic activities but on the other, it can promote tumor progression through different mechanisms such as the induction of vascular endothelial adhesion receptors and the promotion of growth, invasion, and metastasis. The ultimate effect of TNF-␣ may depend on its concentrations, on tumor cell susceptibility, and on the stage of the disease. 3,4

The proprotein convertases (PCs) are implicated in the activation of various precursor proteins that play an important role in tumor cell metastasis. Here, we report their involvement in the regulation of the metastatic potential of colorectal tumor cells. PC function in the human and murine colon carcinoma cell lines HT-29 and CT-26, respectively, was inhibited using siRNA targeting the PCs furin, PACE4, PC5, and PC7 or by overexpression of the general PC inhibitor α1-antitrypsin Portland (α1-PDX). We found that overexpression of α1-PDX and knockdown of furin expression inhibited processing of IGF-1 receptor and its subsequent activation by IGF-1 to induce IRS-1 and Akt phosphorylation, all important in colon carcinoma metastasis. These data suggest that the PC furin is a major IGF-1 receptor convertase. Expression of α1-PDX reduced the production of TNF-α and IL-1α by human colon carcinoma cells, and incubation of murine liver endothelial cells with conditioned media derived from these cells failed to induce tumor cell adhesion to activated murine endothelial cells, a critical step in metastatic invasion. Furthermore, colon carcinoma cells in which PC activity was inhibited by overexpression of α1-PDX when injected into the portal vein of mice showed a significantly reduced ability to form liver metastases. This suggests that inhibition of PCs is a potentially promising strategy for the prevention of colorectal liver metastasis.

In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription-PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ET(A) and ET(B)) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-kappaB (nuclear factor kappaB) activation blocked MMP-2 production and activity, indicating the involvement of NF-kappaB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ET(A) receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.

The receptor for the type 1 insulin-like growth factor (IGF-I) regulates multiple cellular functions impacting on the metastatic phenotype of tumor cells, including cellular proliferation, anchorage-independent growth, survival, migration, synthesis of the 72-kDa type IV collagenase and invasion. We have used site-directed mutagenesis to generate domain-specific mutants of the receptor ␤ subunit to analyze the role of specific tyrosines in the regulation of the invasive/metastatic phenotype. Poorly invasive M-27 carcinoma cells expressing low receptor numbers were transfected with a plasmid vector expressing IGF-I receptor cDNA in which single or multiple tyrosine codons in the kinase domain, namely Tyr-1131, Tyr-1135, and Tyr-1136 or the C-terminal tyrosines 1250 and 1251 were substituted with phenylalanine. Changes in the invasive and metastatic properties were analyzed relative to M-27 cells expressing the wild type receptor. We found that cells expressing the Y1131F,Y1135F,Y1136F or Y1135F receptor mutants lost all IGF-IR-dependent functions and their phenotypes were indistinguishable from, or suppressed relative to, the parent line. The Y1250F,Y1251F substitution abolished anchorage-independent growth, cell spreading, and the anti-apoptotic effect of IGF-I whereas all other IGF-IR-dependent phenotypes were either unperturbed (i.e. mitogenicity) or only partially reduced (migration and invasion). The results identify three types of receptor-dependent functions in this model: those dependent only on an intact kinase domain (DNA synthesis), those dependent equally on kinase domain and Tyr-1250/1251 signaling (e.g. apoptosis, soft agar cloning) and those dependent on kinase domain and enhanced through Tyr-1250/1251 signaling (migration, invasion). They suggest that signals derived from both regions of the receptor cooperate to enhance tumor metastasis.Cancer metastasis is a complex multistep process involving sequential interactions between the disseminating tumor cells and a continuously changing host microenvironment. These interactions include cell-cell and cell-extracellular matrix communication mediated through specific adhesion receptors, such as integrins, which regulate tumor cell attachment, spreading, and migration (1, 2), the dissolution of tissue barriers through the degradative activity of enzymes such as metallo-and serine-proteinases (3, 4) and growth modulation by paracrine, host-derived peptide growth factors (5, 6).The human receptor for the type 1 insulin-like growth factor (IGF-IR) 1 is a heterotetramer consisting of two 130-to 135-kDa ␣ and two 90-to 95-kDa ␤ subunits with several ␣-␣ and ␣-␤ disulfide bridges (7). It is synthesized as a single polypeptide chain, which is then glycosylated and proteolytically cleaved into the ␣ and ␤ subunits (8). The ligand-binding domain is located on the extracellular ␣ subunit. Approximately one-third of the ␤ subunit is extracellular and is connected to the intracellular portion by a single transmembrane domain. The intracellular region of the ␤ subuni...

No abstract