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Furin is the first discovered proprotein convertase member and is present in almost all mammalian cells. Therefore, by regulating the maturation of a wide range of proproteins, Furin expression and/or activity is involved in various physiological and pathophysiological processes ranging from embryonic development to carcinogenesis. Since many of these protein precursors are involved in initiating and maintaining the hallmarks of cancer, Furin has been proposed as a potential target for treating several human cancers. In contrast, other studies have revealed that some types of cancer do not benefit from Furin inhibition. Therefore, understanding the heterogeneous functions of Furin in cancer will provide important insights into the design of effective strategies targeting Furin in cancer treatment. Here, we present recent advances in understanding how Furin expression and activity are regulated in cancer cells and their influences on the activity of Furin substrates in carcinogenesis. Furthermore, we discuss how Furin represses tumorigenic properties of several cancer cells and why Furin inhibition leads to aggressive phenotypes in other tumors. Finally, we summarize the clinical applications of Furin inhibition in treating human cancers.

This article belongs to the Special Issue Calcium Signaling Remodeling and Functional Role in Cancer Cells, [Simple Summary] Furin, a proprotein convertase that belongs to a family of Ca2+-dependent serine peptidases, is involved in the maturation of a variety of proproteins, including growth factors, receptors and differentiation factors, adhesion molecules and proteases. Furin have been associated with tumorigenesis and tumor progression and metastasis; therefore, it has been hypothesized that Furin may constitute a new potential target for cancer therapy. In triple negative breast cancer cells, inhibition of Furin by the prodomain ppFurin results in enhancement of Ca2+ influx, which involves both the increase of store-operated calcium entry (SOCE) and the activation of constitutive Ca2+ entry. The latter involves the activation of Orai and TRPC6 channels, while the increase of SOCE observed in ppFurin-expressing cells is entirely dependent on Orai channels. As a result, ppFurin expression reduces triple negative breast cancer cell viability and ability to migrate and enhances their sensitization to hydrogen peroxide-induced apoptosis., [Abstract] The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells’ malignant phenotype repression and reduction of their resistance to treatments., This research was funded by SIRIC-Brio, La Ligue Contre le Cancer and Region Nouvelle Aquitaine to A.M.K. and by PID2019-104084GB-C21 and PID2019-104084GB-C22/AEI/10.13039/501100011033, MICINN (Grant BFU2016-74932-C2) and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB16046 and GR18061) to J.A.R. and T.S. J.J.L. is supported by a contract from Junta de Extremadura (TA18011). C.C. is supported by a contract from Junta de Extremadura—FEDER.

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains furin-like cleavage sites absent the other SARS-like viruses. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In this study, we used structure- based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti-fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS- CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

Simple Summary Triple-negative breast cancer (TNBC) is known to have a poor prognosis and limited treatment options. The aim of the current study is to evaluate the role of Furin, a proprotein convertase involved in the activation of wide range of protein precursors in TNBC progression. The generation of a TNBC mouse model lacking Furin specifically in the mammary gland confirmed that Furin is implicated in TNBC tumor progression and the derived lung metastasis. Further analysis revealed that the proteolytic activation of proIGF1R and proIR receptors, two substrates of Furin involved in TNBC were inhibited in these mice and was associated with reduced AKT and ERK1/2 expression and phosphorylation. In addition, Furin is frequently overexpressed in TNBC tumors and correlates with poor patient prognosis, suggesting the use of Furin inhibition as a potential adjunct therapy in TNBC. Abstract In triple negative breast cancer (TNBC) cell lines, the proprotein convertase Furin cleaves and then activates several protein precursors involved in oncogenesis. However, the in vivo role of Furin in the mammary gland and how mammary gland-specific Furin knockout specifically influences tumor initiation and progression of TNBC is unknown. Here, we report that Furin is frequently overexpressed in TNBC tumors and this correlates with poor prognosis in patients with TNBC tumors. In a whey acidic protein (WAP)-induced mammary epithelial cell-specific Furin knockout mouse model, mice show normal mammary development. However, loss of Furin in mammary glands inhibits primary tumor growth and lung metastasis in an oncogene-induced TNBC mouse model. Further analysis of TNBC mice lacking Furin revealed repressed maturation of the Furin substrates proIGF1R and proIR that are associated with reduced expression and activation of their downstream effectors PI3K/AKT and MAPK/ERK1/2. In addition, these tissues showed enhanced apoptotic signaling. In conclusion, our findings reveal that upregulated Furin expression reflects the poor prognosis of TNBC patients and highlights the therapeutic potential of inhibiting Furin in TNBC tumors.

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we show that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced SOCE through TRPC6 activation that associated reduced cells malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments, revealed that the ppFurin-expressing cells are unable to process adequately the PC substrates proVEGF-C and proIGF-1R. Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 significantly induces Ca2+ entry which is impaired by silencing of TRPC6 expression. Analysis of TRPC6 activation revealed its up-regulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. The expression of ppFurin in MDA-MB-231 cells reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization leading to breast cancer cells malignant phenotype repression and reduction of their resistance to treatments.

Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment., This work was supported by INSERM, SIRIC Brio, Region Nouvelle Aquitaine, and La Ligue Contre le Cancer.

Immunotherapeutic interventions have become an important treatment for various cancer types including triple negative breast cancer (TNBC). Previous studies have shown that T cell-specific Furin deficient mice show regulatory CD4+ T cells (Tregs) malfunction phenotypes due to impaired cleavage of proTGF-β1. However, it is unknown how this phenotype influences tumor initiation and progression in TNBC. Here, we first show that there is a higher level of Furin expression in different immune cells compared to other proprotein convertase members, and its expression is clearly upregulated once immune cells are activated. Moreover, Furin expression levels negatively correlated with an abundance of different infiltrating immune cells in TNBC tumor samples. In an oncogene-induced TNBC mouse model, we demonstrate that Furin inactivation in T cells inhibits primary tumor growth and lung metastasis. Disruption of Furin in T cells in these mice led to a decreased peripheral and tumor-infiltrating Tregs. As a consequence, tumor-infiltrating CD8+ T cells showed a strong proliferative capacity and upregulated expression of IFN-γ and TNF-α. In these mice the repressed tumor growth was associated with induced apoptosis, which led to reduced lung metastases formation. Taken together, these finding revealed the potential therapeutic benefit of targeting Furin in cancer, particularly for immunotherapeutic interventions to treat TNBC. ispartof: CANCER LETTERS vol:484 pages:40-49 ispartof: location:Ireland status: published

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-β1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment. ispartof: ONCOGENE vol:39 issue:17 pages:3571-3587 ispartof: location:England status: published

Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. In vivo, inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. Significance: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer.

Cartilage loss in osteoarthritis (OA) results from altered local production of growth factors and metalloproteases (MMPs). Furin, an enzyme involved in the protein maturation of MMPs, might regulate chondrocyte function. Here, we tested the effect of furin on chondrocyte catabolism and the development of OA. In primary chondrocytes, furin reduced the expression of MMP-13, which was reversed by treatment with the furin inhibitor α1-PDX. Furin also promoted the activation of Smad3 signaling, whereas activin receptor-like kinase 5 (ALK5) knockdown mitigated the effects of furin on MMP-13 expression. Mice underwent destabilization of the medial meniscus (DMM) to induce OA, then received furin (1 U/mice), α1-PDX (14 µg/mice) or vehicle. In mice with DMM, the OA score was lower with furin than vehicle treatment (6.42 ± 0.75 vs 9.16 ± 0.6, p

Proprotein convertases are subtilisin-like serine endoproteases that cleave and hence activate a variety of proproteins, including growth factors, receptors, metalloproteases, and extracellular matrix proteins. Therefore, it has been suggested that inhibition of the ubiquitously expressed proprotein convertase FURIN might be a good therapeutic strategy for several tumor types. Whether this is also the case for hepatocellular carcinoma (HCC) is currently not clear. In a mouse model for HCC expression ofFurinwas not altered in the tumors, while those of PC7, PC5/6, and PACE4 significantly decreased, at least at some time points. To investigate the impact ofFurininhibition on the development and progression of HCC in this model,Furinwas genetically ablated in the liver.Furininactivation resulted in an increased tumor mass after 5 weeks. This was not caused by decreased apoptosis, since no differences in the apoptosis index could be observed. However, it could at least partially be explained by increased hepatocyte proliferation at 5 weeks. The tumors of theFurinknockout mice were histologically similar to those in wild type mice. In conclusion, liver-specificFurininhibition in HCC enhances the tumor formation and will not be a good therapeutic strategy for this tumor type.

Download Free Sample The vascular endothelial growth factor (VEGF) family members that include VEGF-A, -B, -C, -D, and placental growth factor (PlGF), display distinct binding affinities for their receptors VEGFR-1, -2, and/or -3. In addition to their requirements in the initiation, development, and maintenance of blood and lymphatic vasculature, VEGFs and VEGFRs are upregulated during neoplasia and are involved in the remodeling of tumoral blood and lymphatic vasculature. By activating VEGFR-1 and VEGFR-2, both expressed on blood endothelial cells, VEGF-A promotes the formation of new tumoral blood vessels and thereby accelerates tumor growth. In contrast, upregulation of VEGF-C, a ligand for lymphatic endothelial VEGFR-3 as well as for VEGFR-2, induces the formation of tumor-associated lymphatic vessels and thus promotes the passive metastatic dissemination of tumor cells to regional lymph nodes. Of the VEGF family members, only VEGF-C and -D were found to be proteolytically processed by Furin-...

Proprotein convertases (PCs) are involved in various physiological and pathological processes ranging from embryogenesis to carcinogenesis. Here, using the zebrafish fin regeneration model, we report induced expression of furin and PC5 but not PACE4 and PC7 during fin regeneration that is associated with increased PC activity. Stabilization of HIF by cobalt chloride (CoCl2) further increases these processes. The use of the general PC-inhibitor decanoyl-RVKR-cholromethyl ketone (CMK) inhibited control and CoCl2-induced PC activity. CoCl2 inhibits embryonic zebrafish ZF4 cell proliferation and caudal fin regeneration that is associated with the expression of the anti-proliferative genes P21, P16, PC3 and P53 in ZF4 cells and in non-regenerating stump tissues. In contrast, during fin regeneration, HIF stabilization failed to promote the expression of these anti-proliferative genes and maintained high expression of cyclin D. Further analysis revealed that CoCl2 maintained the formation of immature regenerating vasculature that was associated with amplified expression of OCT4 and various angiogenic factors reported to be PC substrates and/or downstream effectors. These findings revealed that while furin and PC5 expression/activity and their substrates/effectors are regulated during fin regeneration, HIF stabilization by CoCl2 has the potential to modulate these processes and impact on the regenerative process and vessels organization.

Objective Rheumatoid arthritis (RA) is an autoimmune joint disease associated with chronic inflammation of the synovium that causes profound damage of joints. Inflammation results in part from the influx of immune cells secreting inflammatory cytokines and the reduction in the number of Treg cells. We undertook this study to assess the effect of furin, a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of both proteinase maturation and immune cells, in an experimental model of RA. Methods The effect of furin and its inhibitor α1-PDX was tested in mice with collagen-induced arthritis (CIA). Joints were processed for histology and protein expression. Levels of cytokines were measured in joint tissue, and Treg cell numbers were measured in spleens. Results Furin expression and activity were high in the synovial pannus in RA patients and mice with CIA. Systemic administration of furin prevented increases in the arthritis score, joint destruction, and bone loss, in contrast to systemic administration of the furin inhibitor α1-PDX, which enhanced these parameters. By preventing the development of synovial pannus, furin reduced the expression of metalloproteinases in the joints. In contrast, α1-PDX enhanced synovial proliferation and the expression and activity of matrix metalloproteinases. Furthermore, furin reversed the local Th1/Th2 balance and restored the number of Treg cells in the spleen, indicating mediation by immune cells. Conclusion These findings show the protective role of exogenous furin against RA, mediated by an immune response. The data suggest the potential therapeutic use of furin or its derivatives in autoimmune diseases including RA.

Hepcidin is an iron homoeostasis regulator peptide. Loss-of-function mutations cause juvenile haemochromatosis while its over-expression results in anaemia. However, the mechanism and function of preprohepcidin conversion to mature hepcidins (25, 22 and 20 amino acid C-terminal peptides) are not well known. After removal of the signal peptide, the first proteolytic cleavage occurs within the basic motif RRRRR(59)DT, suggesting the involvement of proprotein convertase (PC) family members in this process.Using cell transfection experiments, the processing of preprohepcidin in the human hepatocyte line Huh-7 was found to be inhibited by the Furin inhibitors serpin alpha1-antitrypsin (alpha1-PDX) and prosegment preproFurin (ppFurin). Site-directed mutagenesis analysis confirmed the RRRRR(59)DT preprohepcidin cleavage site. In parallel, the lack of preprohepcidin processing found in the PC activity-deficient cell line LoVo was restored by the expression of Furin, paired basic amino acid cleaving enzyme 4 (PACE4), PC5 or PC7. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of preprohepcidin. In addition, during mouse embryonic development the major expression of hepcidin found in the liver coincided with that of Furin. While hepcidin induces the degradation of the iron transporter ferroportin, its RRRRR(59) to SSSSS(59) mutant is not active.These results demonstrate the key role of the convertases Furin, PACE4, PC5 and/or PC7 in the generation and secretion of active hepcidin and suggest that the control of hepcidin processing as a potential therapeutic/diagnostic strategy in hepcidin-related disorders such as haemochromatosis, inflammatory diseases, anaemia and cancer.

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 alpha1-antitrypsin Portland (alpha1-PDX). We found that overexpression of alpha1-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 alpha1-PDX reduced the production of TNF-alpha and IL-1alpha 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 alpha1-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.

Platelet-derived growth factor-B (PDGF-B) is important for normal tissue growth and maintenance and its overexpression has been linked to several diseases, including cancer, fibrotic disease and atherosclerosis. Here, we show that synthesized as a precursor, proPDGF-B is converted to a mature form by proteolytic cleavage at two sites and its N-terminal cleavage is a prerequisite for processing at its C-terminus. The first cleavage occurs at residues RGRR81/, and the second cleavage close to residues ARPVT190, just before the C-terminal amino-acid sequence crucial for PDGF-B retention to cell surface. Cotransfection of a Furin-deficient cell line LoVo-C5 with proPDGF-B and different PC members revealed that Furin, PACE4, PC5, and PC7 are candidate proPDGF-B convertases. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of proPDGF-B. The processing of proPDGF-B is blocked by site-directed mutagenesis of the RGRR81/ sequence and by various PC inhibitors. Mutation of the PDGF-A and/or PDGF-B convertase sites, revealed that processing of both A and B chains is required for the formation of mature PDGF-B dimers and that the processing of the B chain controls the level of secreted and matrix-bound PDGF-BB forms. Our findings emphasize the importance of the convertase-directed processing of proPDGF-B at the RGRR81/ sequence for PDGF-B maturation and secretion.

Liver is the most common site of metastasis from colorectal cancers, and liver of patients with liver colorectal metastasis have abnormal levels of the proprotein convertases (PCs). These proteases are involved in the activation and/or expression of various colon cancer-related mediators, making them promising targets in colorectal liver metastasis therapy. Here, we revealed that the serpin Spn4 from Drosophila melanogaster inhibits the activity of all the PCs found in the constitutive secretory pathway and represses the metastatic potential of the colon cancer cells HT-29 and CT-26. In these cells, Spn4A inhibited the processing of the PCs substrates IGF-1R and PDGF-A that associated their reduced anchorage-independent growth, invasiveness and survival in response to apoptotic agents. In vivo, Spn4A-expressing tumor cells showed repressed subcutaneous tumor development and liver metastases formation in response to their intrasplenic inoculation. In these cells Spn4A induced the expression of molecules with anti-metastatic functions and inhibited expression of pro-tumorigenic molecules. Taken together, our findings identify Spn4A as the only endogenous inhibitor of all the constitutive secretory pathway PCs, which is able to repress the metastatic potential of colon cancer cells. These results suggest the potential use of Spn4A and/or derivates as a useful adduct colorectal liver metastasis prevention.

Proprotein convertases (PCs) of the subtilisin/kexin family are responsible for the activation of prohormones, protrophic factors, and their receptors. We sought to determine whether loss of PC-mediated activities might affect the malignant phenotypes of cancer cells. Stable transfectants of alpha(1)-antitrypsin Portland (alpha(1)-PDX) cDNA, coding for a potent PC inhibitor, were analyzed in model HT-29 cells (HT-29/PDX) and in other cell lines. Expression of alpha(1)-PDX resulted in a proinsulin-like growth factor-1 receptor (pro-IGF-1R) processing blockade, hence inhibiting the ability of exogenous IGF-1 to induce tyrosine phosphorylation of its beta-subunit and insulin-related substrate-1. Coexpression of IGF-1R with four different PCs or the novel convertase SKI-1 in the furin-defective LoVo-C5 cells demonstrated that pro-IGF-1R ( approximately 200 kDa) cleavage into IGF-1R (beta-subunit, approximately 105 kDa) can be achieved by furin and PC5A, but not by PACE4, PC7, or SKI-1. Expression of alpha(1)-PDX resulted in reduction of DNA synthesis and in anchorage-independent growth. Following serum deprivation, the alpha(1)-PDX transfectants exhibited an enhanced apoptotic phenotype and were insensitive to IGF-1-mediated [(3)H]thymidine incorporation and protection against apoptosis. These cells showed reduced invasiveness that paralleled decreased mRNA levels of urokinase-type plasminogen activator and its receptor, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. Comparative subcutaneous inoculation of cells in nude mice revealed that animals injected with HT-29/PDX cells exhibited delayed and lower incidence of tumor development as well as reduced tumor size. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells, revealed reduced HT-29/PDX tumor vascularization. These findings indicate that PCs actively contribute to the growth and malignant phenotypes of HT-29 tumors, suggesting that PC inhibition strategies may be a useful adduct to the arsenal of colorectal anticancer gene therapies.

Proteolytic maturation of various precursor proteins by the proprotein convertase Furin is now considered as a crucial step in tumor progression and metastasis. Here, we report the repression of the malignant and metastatic potential of carcinoma cells by the prodomain region of Furin (ppFurin), a naturally occurring inhibitor of this convertase. Overexpression of ppFurin in carcinoma cells in a stable manner significantly reduced their convertase activity and ability to mediate processing of the Furin cancer-related substrates platelet-derived growth factor (PDGF)-A and insulin-like growth factor-I receptor precursors. Unprocessed platelet-derived growth factor-A produced by ppFurin expressing cells failed to induce the activation of Akt in the platelet-derived growth factor receptor-expressing cells NIH BALB/c-3T3 and treatment of ppFurin expressing cells with insulin-like growth factor-I failed to induce Akt phosphorylation, compared with controls. The malignant potential of ppFurin expressing cells was significantly reduced as revealed by the loss of anchorage-independent growth and survival that associated their increased chemosensitivity. In vivo, comparative studies revealed that expression of ppFurin in the carcinoma cells MDA-MB-231 and CT-26 cells inhibited tumor growth when subcutaneously inoculated in nude mice. The use of an experimental liver colorectal metastasis model revealed the reduced ability of metastatic carcinoma CT-26 cells to colonize the liver in response to intrasplenic/portal inoculation. Further analyses revealed reduced Furin activity in tumors derived from intrasplenic inoculated mice with ppFurin expressing CT-26 cells. This finding highlights the role of Furin in the malignant and metastatic potential of tumor cells and suggests the possible consideration of using its naturally occurring inhibitor ppFurin in anticancer therapy.

The proprotein convertases (PCSKs) are involved in the proteolytic maturation/activation of a wide range of protein precursors involved in neoplasia such as adhesion molecules, growth factors, growth factor receptors, and metalloproteinases. Expression analysis of all the PCSKs family members, namely PC1, PC2, furin, PC4, PC5, PACE4, and PC7 in various human and murine melanoma cells revealed increased PCSK6/PAECE4 expression while compared to melanocytes. The use of in vitro digestion assays and cell transfection experiments revealed that targeting the PCSK6/PAECE4 in melanoma cells using small interfering RNA (siRNA) reduced PCSKs activity and repressed the processing the PCSKs substrates proIGF-1R, pro-VEGF-C and proPDGF-A. Theses unprocessed substrates failed to mediate their signaling pathways that associated reduced cell proliferation. Furthermore, PCSK6/PAECE4 gene silencing reduced melanoma cells migration and invasion that paralleled decreased gelatinase MMP-2 and MMP-9 activity and altered expression and secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Taken together, these findings highlight the importance of PCSK6/PAECE4 activity in melanoma cells and suggest PCSK6/PAECE4 inhibition as a potentially promising strategy for the prevention of melanoma invasiveness. Citation Format: Geraldine Siegfried, Apolline Imbard, Serge Evrard, Abdel-Majid Khatib. Targeting the proprotein convertase PCSK6/PAECE4 abrogates human melanoma malignant phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 696.

Aim of the study: Cartilage degradation in OA is mainly due to a reduced matrix protein secretion and an increase of their degradation by matrix metalloprotease (MMP). Indeed during OA an enhanced expression and activation of MMPs is observed. The major MMPs involve in matrix protein degradation are ADAMTS-4, MMP-3 or MMP-13. However, inhibition of expression or activation of MMPs might reduce matrix degradation during OA. Furin, a proprotein convertase, is responsible for the conversion of various Pro-MMPs in active MMPs. Furin activates directly ADAMTS-4 and MMP-3, while promote indirectly the maturation of MMP-13 through other MMPs. Therefore, we hypothesised that Furin may have an impact in matrix homeostasis in joint instability. Moreover, we investigated the in vitro effect of Furin in MMP expression in murin chondrocytes. Methods: DMM (destabilisation medial meniscus) was induced surgically by partial meniscectomy in 10-week-old C57Bl6 male mice. The day after surgery, the mice received twice weekly intraperitoneal injections of Furin (1U/mouse), or a1-PDX (the proprotein convertase inhibitor, 0.35 mg/g mice), or vehicle (control group) and sacrificed after 6 weeks. Joint sections were used to assess cartilage damage (OARSI score) and the number of ADAMTS-4 and MMP-13(+) cells as well as Furin expressing cells. We further assessed the signalling pathway induced by Furin in vitro. Murine primary chondrocytes (pre-treated with 10ng/ml IL-1) were cultured with Furin (10U/mL) or a1-PDX (8mM) for 48h and then analyzed the expression of MMP-13 and the phosphorylation of Smad2/3 in cell lysates by Western blot. Results:We first observed a constitutive expression of Furin in cartilage of control mice while absent in DMM mice. Furin reduces OA score (5.64 1.54) but unchangedwith a1-PDX (15.16 2.82) compared to vehicle (13.92 1.95, p

Processing of cancer-associated precursor proteins such as growth factors by the Proprotein Convertase furin is an important cellular process for acquisition of malignant phenotype and metastatic potential of tumor cells. Furin is inhibited by its own prodomain protein which also plays role in the folding of mature furin. Herein, the complete 83-mer furin prodomain protein was chemically synthesized for the first time by solid phase peptide chemistry and its effects on furin activity and tumor cells malignant phenotypes were evaluated. It inhibited furin activity in a competitive manner with low nanomolar inhibition constant (Ki). Expression of furin prodomain cDNA in tumor cells or cells incubated with the corresponding protein blocked furin-cleavage of proPDGF-A. This was associated with significant reduction in tumor cells proliferation, migration as well as invasion. Interestingly shorter synthetic furin prodomain peptides derived from either its primary or secondary activation site alone weakly inhibited furin and displayed limited effects on tumor cells. This suggests that the combined presence of the above two prodomain segments is crucial for prodomain's potent furin-inhibitory and hence anticancer activities.

Proteolytic cleavage of various cancer-related substrates by the proprotein convertases (PC) was reported to be important in the processes of neoplasia. These enzymes are inhibited by their naturally occurring inhibitors, the prosegments (ppPC), and by the engineered general PC inhibitor, the serpin variant α1-PDX. In the present study, we sought to compare the effect of these PC inhibitors on malignant phenotypes of breast cancer cells. Overexpression in a stable manner of α1-PDX and the prosegment ppPACE4 in MDA-MB-231 breast cancer cells resulted in increased matrix metalloproteinase (MMP)-9 (but not MMP-2) activity and a reduced secretion of tissue inhibitor of metalloproteinase 1 (TIMP-1). This was associated with significant enhancement in cell motility, migration, and invasion of collagen in vitro. In contrast, ppFurin expression in these cells decreased MMP-9 activity and diminished these biological functions, but had no significant effect on TIMP-1 secretion. Taken together, these data showed the specific and opposing roles of Furin and PACE4 in the regulation of MMP-9/TIMP-1–mediated cell motility and invasion. [Cancer Res 2007;67(19):9030–4]

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 proprotein convertases represent a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7, and two other subtilases that cleave at non-basic residues, called SKI-1/S1P and NARC-1/PCSK9. The present review concentrates on the regulatory role played by some of these convertases in cholesterol and lipid metabolism. Thus, PC5/6, PACE4 and Furin upregulate high-density lipoprotein (HDL) levels via the inactivation of endothelial and lipoprotein lipases. The SKI-1/S1P-directed cleavage of membrane-bound transcription factors known as sterol regulatory element binding proteins (SREBP-1 and SREBP-2) results in upregulation of the synthesis of sterols, lipids and the LDL receptor (LDLR). Finally, PCSK9 downregulates the protein levels of the LDLR by enhancement of its intracellular metabolic pathway in subcellular acidic compartments.

The members of the convertase family play a central role in the processing of various protein precursors ranging from hormones and growth factors to viral envelope proteins and bacterial toxins. The proteolysis of these precursors that occurs at basic residues is mediated by the proprotein convertases (PCs), namely: PC1, PC2, Furin, PACE4, PC4, PC5 and PC7. The proteolysis at non-basic residues is performed by subtilisin/kexin-like isozyme-1 (S1P/SKI-1) and the newly identified neural apoptosis-regulated convertase-1 (NARC-1/PCSK9). These proteases have key roles in many physiological processes and various pathologies including cancer, obesity, diabetes, neurodegenerative diseases and autosomal dominant hypercholesterolermia. Here we summarize the discovery of the proprotein convertases and their inhibitors, discuss their properties, roles, resemblance and differences

The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR(227)SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C ((220)Q-VHSIIRR downward arrow SLP(230)). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR(227)SL to HSIISS(227)SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.

Although altered expression of platelet-derived growth factor (PDGF)-A is a hallmark of many cancers, the importance of pro-PDGF-A conversion to PDGF-A in tumorigenesis and the cognate protease(s) is unknown. Pro-PDGF-A processing occurs at pairs of basic residues, likely involving the proprotein convertases (PCs). In the colon carcinoma cell line LoVo, we found that Furin is the most potent PDGF-A convertase. Mutation of the PC-site RRKR(86) to ARKA(86) inhibited pro-PDGF-A processing, its receptor tyrosine phosphorylation, and cell proliferation. This processing is also blocked by the PC preprosegments (pps) ppFurin, ppPC5, and ppPACE4, and by the Furin-variants of alpha2-macroglobulin and alpha1-antitrypsin. Chinese hamster ovary cells overexpressing pro-PDGF-A (ARKA(86)) failed to induce tumors in nude mice. Thus, PC-directed inhibitors might represent new agents for therapy in neoplasia induced by PDGF-A.

Background In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR(214) suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration. Methodology/principal findings Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR(214) into HSIISS(214)). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4-7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration. Conclusions/significances Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration.

Background: Furin represents a crucial member of secretory mammalian subtilase, the Proprotein Convertase (PC) or Proprotein Convertase Subtilisin/Kexin (PCSK) superfamily. It has been linked to cancer, tumorgenesis, viral and bacterial pathogenesis. As a result it is considered a major target for intervention of these diseases. Methodology/Principal Findings: Herein, we report, for the first time, the synthesis and biological evaluation of a newly designed potent furin inhibitor that contains a highly reactive beta-turn inducing and radical generating ‘‘enediynyl amino acid’’ (Eda) moiety. ‘‘Eda’’ was inserted between P1 and P19 residues of hfurin 98–112 peptide, derived from the primary cleavage site of furin’s own prodomain. The resulting hexadecapeptide derivative inhibited furin in vitro with IC50 ,40 nM when measured against the fluorogenic substrate Boc-RVRR-MCA. It also inhibited furin-mediated cleavage of a fluorogenic peptide derived from hSARS-CoV spike protein with IC50 ,193 nM. Additionally it also blocked furin-processing of growth factors proPDGF-A, B and VEGF-C that are linked to tumor genesis and cancer. Circular dichroism study showed that this inhibitor displayed a predominantly beta-turn structure while western blots confirmed its ability to protect furin protein from self degradation. Conclusion/Significance: These findings imply its potential as a therapeutic agent for intervention of cancer and other furin-associated diseases.

The members of the proprotein convertase family play a central role in the processing and/or activation of various protein precursors involved in many physiological processes and various pathologies. The proteolysis of these precursors that occur at basic residues within the general motif (K/R)-(X)-(K/R) is mediated by the proprotein convertases PC1/3, PC2, Furin, PACE4, PC4, PC5 and PC7, whereas the proteolysis of precursors within hydrophobic residues performed by the convertase S1P/SKI-1 and the convertase NARC-1/PCSK9 seems to prefer cleavages at the motif LVFAQSIP. Here we provide a comprehensive overview of their remarkable complex roles as revealed by disruption of their genes individually using generalized or conditional approaches.