Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.     

Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-beta (TGFbeta)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFbeta-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFbeta-related factors' biological activity.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix: Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-β (TGFβ)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFβ-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFβ-related factors' biological activity.     

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.     

The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2

PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.     

Development of selectivity of alpha1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crucial role of the P4 arginine in PACE4 inhibition

PACE4, furin and PC6 are Ca2+-dependent serine endoproteases that belong to the subtilisin-like proprotein convertase (SPC) family. Recent reports have supported the involvement of these enzymes in processing of growth/differentiation factors, viral replication, activation of bacterial toxins and tumorigenesis, indicating that these enzymes are a fascinating target for therapeutic agents. In this work, we evaluated the sensitivity and selectivity of three rat alpha1-antitrypsin variants which contained RVPR352, AVRR352 and RVRR352, respectively, within their reactive site loop using both inhibition of enzyme activity toward a fluorogenic substrate in vitro and formation of a SDS-stable protease/inhibitor complex ex vivo. The RVPR variant showed relatively broad selectivity, whereas the AVRR and RVRR variants were more selective than the RVPR variant. The AVRR variant inhibited furin and PC6 but not PACE4. This selectivity was further confirmed by complex formation and inhibition of pro-complement C3 processing. On the other hand, although the RVRR variant inhibited both PACE4 and furin effectively, it needed a 600-fold higher concentration than the RVPR variant to inhibit PC6 in vitro. These inhibitors will be useful tools in helping us to understand the roles of PACE4, furin and PC6.     

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.     

The activation and physiological functions of the proprotein convertases

The mammalian secretory proprotein convertases are part of a family of nine serine proteinases of the subtilisin-type. Seven of them cleave after basic amino acids and are called PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7. The two other convertases SKI-1/S1P and PCSK9 are implicated in cholesterol and/or fatty acid metabolism. The convertases PC5/6 and PACE4 are activated at the cell surface where they are tethered to heparan sulfate proteoglycans. This activation pathway is unique and differs from that of furin and PC7, which are activated in the trans-Golgi network and from PC1/3 and PC2 that are activated in dense core secretory granules. While some of the basic amino acid-specific convertases may display redundant cleavages of substrates, they uniquely process certain substrates in vivo. Indeed, the conditional knockout of the PC5/6 gene in the embryo proper in mice led to severe malformations, bone morphogenic defects and death at birth. This is likely due to the absence of processing of the growth differentiating factor 11 (Gdf11). Both complete and liver-specific knockout of Pcsk9 revealed that it is a major convertase that regulates the level of circulating low-density lipoproteins (LDL) via the degradation of the hepatic LDL-receptor. This apparently non-enzymatic mechanism implicates the enhanced degradation of the LDLR in endosomes/lysosomes. These data provide evidence that an inhibitor of PCSK9-LDLR interaction is a viable target for the development of a novel cholesterol lowering drug in conjunction with the classical statins.     

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.     

Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2´ inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2´ of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1) mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2) two proprotein convertases (PC1 and PC2) may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5) are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7) compared to the remaining two (PC1 and PC2). The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein convertases and the development of therapeutic applications.     

The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates

The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

Proprotein convertases in post-menopausal endometrial cancer: distinctive regulation and non-invasive diagnosis

Proprotein convertases (PCs) play critical roles in cleaving precursor proteins (growth factors, hormones, receptors and adhesion molecules) for activation. PCs are implicated in a number of cellular functions, including oncogenesis. Endometrial cancer is the most common gynecological cancer in the developed world, but the involvement of PCs is unclear. To characterize the role of PCs in endometrial cancer, we assessed expression of seven PCs (PC1/3, PC2, PACE4, PC4, furin, PC5/6 and PC7) by RT-PCR in six well characterized endometrial cancer cell lines. Expression was variable in all lines, with furin being most consistently expressed in all cell lines tested. We next determined the cellular localization and expression levels of four ubiquitously expressed PCs (furin, PACE4, PC5/6 and PC7) in post-menopausal endometrial biopsies from control (n=7) and endometrial cancer patients (n=30) by immunohistochemistry. Furin increased in tumors, whereas PC5/6, PACE4 and PC7 expression was reduced with increasing cancer grades. Uterine lavage is a non-invasive source material for evaluating the endometrium. We thus assessed whether total PC activity was altered in uterine lavage of endometrial cancer patients (n=36) compared to controls (n=10). PC activity was detected in all uterine lavage samples, and significantly elevated in all grades of endometrial cancer. This study demonstrates a complex association between individual PCs and endometrial cancer. Importantly, we show that monitoring the total PC activity in uterine lavage may provide a rapid and non-invasive method for the diagnosis of endometrial cancer in postmenopausal women.