Pascale Reverdiau

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 VEGF165 binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 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.

Inhibition of cervical cancer cell growth by human papillomavirus virus-like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth. [Mol Cancer Ther 2009;8(2):357–65]

Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion.

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.

Demonstration of a Tissue Factor Pathway Inhibitor 2 Messenger RNA Synthesis by Pure Villous Cytotrophoblast Cells Isolated from Term Human Placentas

Abstract Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.

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 α1 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.

Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor.

Abstract The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor

Abstract Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI‐2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI‐2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI‐2 in tumour progression, TFPI‐2 was overexpressed in NCI‐H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI‐2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI‐H209 cells expressing TFPI‐2. We also demonstrated that TFPI‐2 upregulation in NCI‐H209 cells decreased MMP expression, particularly by downregulating MMP‐1 and MMP‐3. Moreover, TFPI‐2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP‐1 was predominant in SCLC tissues and was inversely expressed with TFPI‐2 in 35% of cases. These results suggest that downregulation of TFPI‐2 expression could favour the development of SCLC.

Effects of Phosphatidic Acid on Cytokinin Signal Transduction in Periwinkle Cells

In periwinkle cell suspensions, the accurate quantification of gene expression through real-time RT-PCR showed that two type-A response regulators (RR-A), considered primary cytokinin (CK)-responsive genes, were differentially regulated after CK treatment. Specific inhibition of phospholipase D (PLD)-dependent phosphatidic acid (PA) production by primary alcohols reduced significantly the transcript level of one gene in response to CK, although the other gene was unaffected. Moreover, this inhibitory effect on gene transcript level could be antagonized by exogenous supply of PA. These results suggest that PA, likely released from the membrane by PLD activity, could operate in the early steps of CK signalling in periwinkle cells.