Tumor cells do not constitutively exhibit invasive activity, but rather, can be transiently induced to adhere and form lesions. We report here that the expression of seprase, a dominant EDTA-resistant gelatinase in malignant tumors, is dependent on tumor cell exposure to type I collagen gel (TICg). The induced seprase expression of ovarian tumor cells influences their collagen contraction and invasion capability. Importantly, tumor cells with reduced seprase expression, due to manipulation by RNA interference, showed a reduction of TICg contraction in the gel contractility assay, inhibition of tumor cell invasion through TICg as shown by a transwell migration assay and inhibition of peritoneal membrane tumor lesion in a mouse model. In addition, mAb C27, an antibody against b1 integrin, which blocks cellular avidity to TICg, can induce seprase RNA expression and promote the invasive phenotype and metastatic potential of ovarian tumor cells. Thus, collagenous matrices in the tumor cell niche induce the expression of seprase and initiate tumor invasion and metastatic cascades. ' 2008 Wiley-Liss, Inc.Key words: seprase; FAP-a; gelatinase; collagen; ovarian tumor Epithelial ovarian cancer progresses by increased tumor cell proliferation and proteolytic activity that degrades the extracellular matrix (ECM) of the peritoneum. 1,2 Although blocking tumor progression by inhibiting protease activity is well-known, it is also possible that tumor progression could be suppressed by hindering the initial induction of these proteases.Type I collagen (TIC) is comprised of 3 strands of gelatin each of which contain adjoining glycine and proline residues. 3 It is this bond that is the known substrate for seprase. 4 Seprase was first described in the LOX human malignant melanoma cell line and is a type II transmembrane, 760 aa glycoprotein whose 97 kDa monomer homodimerizes to form a catalytically active complex of 170 kDa. 5-7 Molecular cloning of seprase revealed it to be identical to that of fibroblast activation protein a (FAP-a) in gene sequence, protein form, as well as dipeptidyl peptidase and gelatinase activities, even though FAP-a was independently identified in reactive stromal fibroblasts but not in tumor or endothelial cells. [6][7][8][9][10][11] In fact, seprase was found to be present on tumor cells of gastric, colon, melanoma, ovarian and breast cancers. [5][6][7][12][13][14][15] It is likely that activation, which involves the seprase dimer being shorn from the cell membrane to allow greater accessibility of its TICg substrate to the catalytic pore, is required prior to gelatinase activity. 16,17 Seprase is not only present on activated fibroblast during wound healing and active endothelial cells during angiogenesis, but also has been found to be constitutively expressed at high levels in some malignant melanoma, glioma and carcinoma cells; however, most epithelial tumor cell lines express little or no seprase (see gene expression profiles of NCI60 tumor cell lines http:// cgap.nci.nih.gov/Microarray/FNResults?O...

Among the many proteases associated with human cancer, seprase or fibroblast activation protein A, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTAsensitive activators, r-seprase was converted into 70-to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody-or lectinaffinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion. (Cancer Res 2006; 66(20): 9977-85)

Neurotrophin-3 (NT-3), a member of the neurotrophin family, has been shown to be necessary for the development of muscle spindle and Merkel cell aVerent nerve fibres in animal models.The presence of NT-3 in the suprabasal epidermis, where many unmyelinated sensory fibres terminate, has been shown for the first time. As these fibres are aVected in early diabetic neuropathy and a clinical trial of recombinant human NT-3 in diabetic neuropathy is in progress, the concentrations of endogenous NT-3 in skin of 24 patients at diVerent stages of diabetic polyneuropathy have been investigated. NT-3 concentrations, measured with a specific immunoassay, were significantly higher in aVected skin biopsies from patients with diabetic neuropathy than matched control skin (diabetic skin 6.32 (1.18) pg/mg v control skin 1.28 (0.05) (mean (SEM)); p<0.004, Mann-Whitney U test), particularly in the later stages. The optical density of NT-3-immunostaining was also significantly greater in the epidermis in diabetic patients (diabetic epidermis 0.30 (0.06) v controls 0.24 (0.01); p<0.02). No correlation was found between individual quantitative sensory tests and the increase of NT-3 concentration. The increase of NT-3 seems to reflect the degree of skin denervation in diabetic neuropathy, and may represent a compensatory mechanism. The concentrations of NT-3 in other peripheral targets deserve study in diabetic neuropathy. (J Neurol Neurosurg Psychiatry 1998;65:393-395)

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