No abstract

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with nonsmall-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4 -120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P ¼ 0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P ¼ 0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P ¼ 0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC.

These results suggest that high TF expression in lung tumors may result from K-ras mutation and contribute to NSCLC progression, probably via mechanisms other than angiogenesis.

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-␤1 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.

Delivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.

The proinflammatory chemokine CC chemokine ligand 5 (CCL5) is a potent chemoattractant of immature dendritic cells (iDCs). It remains to be elucidated whether CCL5 may also enhance iDC migration through the basement membrane by affecting matrix metalloproteinase (MMP)-9 secretion. In this study, iDCs were differentiated in vitro from human monocytes of healthy donors. Zymographic analysis of cellular membranes of nontreated iDCs revealed a basal secretion of the pro- and active MMP-9, whereas only pro-MMP-9 was detected in conditioned media. Increasing concentrations of CCL5 significantly enhanced MMP-9 secretion by iDCs, peaking at 100 ng/ml, which optimally increased iDC migration through a reconstituted basement membrane (Matrigel) in vitro. The CCL5-enhanced secretion of MMP-9 occurred early (2 h) and was maintained at least for 10 h. A significant increase in MMP-9 mRNA synthesis was detected by reverse transcriptase-polymerase chain reaction, only at 6 h of CCL5 treatment, which suggests that the early effect of CCL5 (0-4 h) on MMP-9 secretion was independent of mRNA synthesis, whereas the more delayed effect (6-10 h) could be mediated through an increase in MMP-9 gene expression. In a Matrigel migration assay, the CCL5-enhanced iDC migration was reduced significantly by specific inhibitors of MMP-9, such as tissue inhibitor of metalloproteinase-1 or an anti-MMP-9 antibody, which indicates that iDC migration through the basement membrane depends on MMP-9. These results suggest that under inflammatory conditions, the chemokine CCL5 may enhance iDC migration through the basement membrane by rapidly increasing their MMP-9 secretion.

Most housekeeping genes, tumor-suppressor genes, and approx 40% of tissue-specific genes contain G+C sequences in their promoter region that were very difficult to amplify. In this report, we propose an improved polymerase chain reaction (PCR) method to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site. Therefore, this method can be recommended to amplify other GC-rich genomic templates.

Tissue factor pathway Inhibitor-2 (TFPI-2) is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. In this study, a 4.8-kb human TFPI-2 gene 5'-flanking region was isolated, cloned and sequenced. Promoter region analysis revealed a high GC-rich content without canonical TATA and CAAT boxes but three transcription initiation sites were identified. Moreover, several putative binding sites for transcription factors were identified (MyoD, LYF1, NF-Y, GATA, oct-1, AP-1, Sp1, NF1, NF-kappa B and egr-1). To characterize potential regulatory regions, TFPI-2/luciferase promoter constructs were then transfected in human choriocarcinoma JEG-3 cells. We first showed that the minimal TFPI-2 promoter is located between -166 and -111 from the translation start site. Luciferase activity consistently increased after stimulation of JEG-3 cells by phorbol 12-myristate 13-acetate indicating that NF1, NF-kappa B and egr-1/Sp1 binding sites are crucial in inducible TFPI-2 expression. Moreover, negative regulatory regions included AP-1 binding sites were identified. This study demonstrates that the TFPI-2 gene promoter exhibits typical features of a housekeeping gene.