Tissue factor pathway inhibitor 2 is a potent kallikrein-related protease 12 inhibitor.

The protease activities are tightly regulated by inhibitors and dysregulation contribute to pathological processes such as cancer and inflammatory disorders. Tissue factor pathway inhibitor 2 (TFPI-2) is a serine proteases inhibitor, that mainly inhibits plasmin. This protease activated matrix metalloproteases (MMPs) and degraded extracellular matrix. Other serine proteases are implicated in these mechanisms like kallikreins (KLKs). In this study, we identified for the first time that TFPI-2 is a potent inhibitor of KLK5 and 12. Computer modeling showed that the first Kunitz domain of TFPI-2 could interact with residues of KLK12 near the catalytic triad. Furthermore, like plasmin, KLK12 was able to activate proMMP-1 and -3, with no effect on proMMP-9. Thus, the inhibition of KLK12 by TFPI-2 greatly reduced the cascade activation of these MMPs and the cleavage of cysteine-rich 61, a matrix signaling protein. Moreover, when TFPI-2 bound to extracellular matrix, its classical localisation, the KLK12 inhibition was retained. Finally, TFPI-2 was downregulated in human non-small-cell lung tumour tissue as compared with non-affected lung tissue. These data suggest that TFPI-2 is a potent inhibitor of KLK12 and could regulate matrix remodeling and cancer progression mediated by KLK12.

Angiogenesis stimulated by human kallikrein-related peptidase 12 acting via a platelet-derived growth factor B-dependent paracrine pathway.

KLK12, a kallikrein peptidase, is thought to take part in the control of angiogenesis. Our analysis of the secretome of endothelial cells (ECs) that had been treated with KLK12 showed that KLK12 converts the extracellular matrix- or membrane-bound precursor of platelet-derived growth factor B (PDGF-B) into a soluble form. Both PDGF-B and vascular endothelial growth factor A (VEGF-A) take part in the induction of angiogenesis by KLK12 in a coculture model of angiogenesis that mimics endothelial tubule formation. We used a cellular approach to analyze the interplay between KLK12, PDGF-B, and VEGF-A and showed that release of PDGF-B by KLK12 leads to the fibroblast-mediated secretion of VEGF-A. This then stimulates EC differentiation and the formation of capillary tube-like structures. Thus, KLK12 favors the interaction of ECs and stromal cells. The released PDGF-B acts as a paracrine factor that modulates VEGF-A secretion by stromal cells, which ultimately leads to angiogenesis. Moreover, the genes encoding KLK12 and PDGFB are both expressed in ECs and up-regulated in tumor cells kept under hypoxic conditions, which is consistent with the physiological involvement of KLK12 in PDGF-B maturation.

Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors.

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-ß1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.

TFPI-2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour-stromal cell interactions.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α(1) integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.

The chicken miR-150 targets the avian orthologue of the functional zebrafish MYB 3'UTR target site.

BACKGROUND: The c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3'UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different. RESULTS: We identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3'UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3'UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3'UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3'UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3'UTR. CONCLUSION: Our study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals.

MAbDelivery: Administration routes for antibody therapy Third LabEx MAbImprove industrial workshop, July 2, 2015 Tours, France.

The annual "LabEx MAbImprove Industrial Workshops" are primarily intended to provide a comprehensive view about topics of interest for the pharmaceutical industry to scientists involved in research on therapeutic antibodies. The third workshop in this series, held July 2, 2015 in Tours, was dedicated to the optimization of delivery, namely all processes leading monoclonal antibodies to reach their target site. The commonly used intravenous (IV) route, although advantageous in terms of pharmacokinetics and pharmacodynamics, presents some disadvantages in terms of patients' convenience, therapeutic target access or treatment cost. Such problems led pharmaceutical companies to consider more straightforward and patient-friendly administration routes, bringing the need for specific formulations adapted to the specific inherent physicochemical challenges. In this context, the workshop provided an overview of these advances and opened discussion on new administration routes and formulation development. In the first session, the opportunities and challenges of 3 main routes of administration (IV, subcutaneous (SC), and pulmonary) were discussed, integrating protein stability issues. The next session was dedicated to medical devices intended for SC and pulmonary administration. The last session focused on specific formulations for monoclonal antibodies, particularly to successfully protect antibodies upon aerosolization, to develop highly concentrated formulations for SC administration, and to use formulation as a mean to overcome the barriers to oral protein delivery. As in the previous editions, this workshop gathered people from the academic and industrial spheres and allowed rich debates and discussions.

Antibody biosimilars: fears or opportunities?: First LabEx MAbImprove industrial workshop, May 28, 2013; Tours, France.

The annual "LabEx MAbImprove industrial workshops" are primarily intended to provide scientists involved in therapeutic antibodies, a comprehensive view about topics of interest for the pharmaceutical industry. They are organized by the "LabEx MAbImprove industrial committee", for this first edition especially in partnership with ARITT, the regional agency for innovation and technology transfer which operates in the French Région Centre, the 1st French region for pharmaceutical production. The 2013 edition, held May 28 at the Vinci Center of Tours, was dedicated to antibody biosimilars. Depending on opinions, the impending expiry of antibody patents and the imminent marketing approval of competitors to blockbusters can be perceived as good or bad things. Fears or opportunities? Risks for patients? Breath of fresh air for the health systems? Opportunity for re-industrializing France? In this context, it is necessary for people to form a fair and informed opinion on the current landscape of antibody biosimilars. In particular, this is especially important for scientists from the academic world, from the industry or from the regulation agencies, for pharmacists, for pharmacovigilance specialists, for health authorities, and staff from health insurance and decision makers. The first session was devoted to market and regulatory issues, and included both an overview of the evolution of the patent landscape and a description of biosimilars regulation in the European Union (EU). This session was closed by a talk on manufacturing processes for biosimilars. In the next session, quality control attributes of biosimilars were discussed and compared with the consistent quality of biotechnology products to raise the question: "How close is close enough?" In vitro assays for evaluating the Fc function of therapeutic antibodies were also discussed. The third session focused on development of biosimilars and primarily on the stepwise process for introducing an antibody biosimilar on the EU market, and included a presentation of the ongoing clinical evaluation of an infliximab biosimilar. The session concluded with a rich debate on the indication extrapolation of a biosimilar compared to the originator. The last session was dedicated to societal issues and focused on two aspects: (1) the need of biosimilars for EU health economy; and (2) last but not least, the ethical issues about clinical evaluation of biosimilars. All speakers and attendees enjoyed this very stimulating and rewarding meeting, which gathered many people with divergent scientific backgrounds from the academic or industrial world.

Autophagy can be a killer even in apoptosis-competent cells.

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.