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The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice. [ABSTRACT FROM AUTHOR]

Furin, one of the members of the family of proprotein convertases (PCs), ubiquitously expressed as a type I membrane- bound proteinase, activates several proteins that contribute to tumor progression. In vitro studies using cancer cell lines and clinical specimens demonstrated that furin processes important substrates such as insulin-like growth factor 1 receptor (IGF-1R) and transforming growth factor β, leading to increased tumor growth and progression. Despite the numerous studies associating furin with tumor development, its effects in preclinical models has not been comprehensively studied. In this study, we sought to determine the protumorigenic role of furin in vivo after a two-stage chemical carcinogenesis protocol in transgenic mice in which furin expression was targeted to the epidermal basal layer. We found that processing of the PC substrate IGF-1R and the proliferation rate of mouse epidermis was enhanced in transgenic mice when compared with their WT counterparts. Histopathologic diagnoses of the tumors demonstrated that furin transgenic mice (line F47) developed twice as many squamous carcinomas as the control, WT mice (P < .002). Similarly, tumors cells from transgenic mice were able to process PC substrates more efficiently than tumor cells from WT mice. Furthermore, furin expression resulted in a higher SCC volume in transgenic mice as well as an increase in the percentage of high-grade SCC, including poorly differentiated and spindle cell carcinomas. In conclusion, expression of furin in the basal layer of the epidermis increased tumor development and enhanced tumor growth, supporting the consideration of furin as a potential target for cancer treatment. [ABSTRACT FROM AUTHOR]

VILIP-1, a member of the neuronal Ca2+ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5- VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ~49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene. [ABSTRACT FROM AUTHOR]

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5′-aza-2′-deoxycytidine (5′-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients. [ABSTRACT FROM AUTHOR]

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease. [ABSTRACT FROM AUTHOR]

Purpose: Squamous cell carcinoma (SCC) of the tongue is a common malignancy of the oral cavity. Furin convertase activates several precursor matrix metalloproteinases involved in the degradation of the extracellular matrix. The pattern of expression of furin and vascular endothelial growth factor-C (VEGF-C), two key molecules in neoplasm development, was examined during the progression from normal epithelium to invasive SCC., Experimental Design: We evaluated furin and VEGF-C expression and microvessel density (MVD) by immunohistochemistry in human tongue sections harboring normal epithelium, dysplastic epithelium, and/or SCC. Sections from 46 glossectomy specimens were assessed for furin expression. A selected group of 15 cases, each containing normal epithelium, precursor lesions, and invasive SCC, were further studied for furin and VEGF-C expression and MVD quantification. We also evaluated the pattern of furin expression and VEGF-C processing by Western blot analysis in three SCC cell lines with different degrees of aggressiveness., Results: Furin and VEGF-C expression was notably higher in most precursor lesions and SCCs than in normal epithelia. Approximately 60% (n = 26) and 100% (n = 15) of the normal epithelia showed low-intensity staining for furin and VEGF-C, respectively. Intense staining for furin and VEGF-C was detected in approximately 80% (n = 34) and 100% (n = 15) of the SCCs, respectively. A significant correlation was seen between the expression of these two markers (Spearman's test, P < 0.00002). We found a statistically significant increase in MVD when either dysplasia (432 +/- 19.06; P < 0.05) or SCC (546 +/- 17.24) was compared with normal epithelium (315 +/- 17.27; P < 0.0001). SCC71, the most aggressive cell line analyzed, was the one with the highest furin expression. This cell line totally processed the VEGF-C proform, whereas the less aggressive line SCC9, exhibiting the least furin expression, did not. SCC15, of intermediate aggressiveness and furin expression, showed intermediate pro-VEGF-C processing., Conclusions: These findings suggest that furin is a useful marker of tumor progression and is responsible for VEGF-C processing. This in turn would enhance angiogenesis, leading to increased MVD associated with preinvasive and invasive neoplasia.

Visinin-like protein-1 (VILIP-1) is a member of the neuronal EF-hand Ca(2+)-sensor protein family. VILIP-1 is expressed in the central nervous system where it plays a crucial role in regulating cAMP levels, cell signaling, and differentiation. Screening of mouse skin tumor cell lines for differentially expressed genes showed high-level VILIP-1 expression in less aggressive squamous cell carcinoma (SCC) and papilloma cell lines. Conversely, expression was markedly decreased or lost in invasive SCC and spindle cell carcinoma cell lines. In addition, immunohistochemistry of normal skin and primary tumors showed that VILIP-1 is expressed in basal cells of the normal intrafollicular epidermis as well as in basal cells of papillomas. The expression was decreased in low-grade SCCs and disappeared in most high-grade SCCs. When two high-grade carcinoma cell lines were transfected with VILIP1-cDNA, the VILIP-1 transfectants had significantly higher cAMP levels than the respective vector alone-transfected lines. VILIP-1-transfected cells were less invasive (both in vivo and in vitro) than the control transfectants. Reduced invasiveness and elevation of cAMP levels were accompanied by decreased MMP-9, as well as decreased RhoA activity. These results indicate that VILIP-1 plays an important role in regulating tumor cell invasiveness and that its loss could aid in enhancing the advanced malignant phenotype.

Purpose: Astrocytoma arises in the central nervous system as a tumor of great lethality, in part because of the invasive potential of the neoplastic cells that are able to release extracellular matrix-degrading enzymes. Furin convertase activates several precursor matrix metalloproteases involved in the breakdown of the extracellular matrix. In the present study inhibition of furin was achieved by gene transfer of alpha(1)-antitrypsin Portland (PDX) cDNA., Experimental Design: This furin inhibitor was transfected into two tumorigenic astrocytoma cell lines. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, and proliferation assays, as well as by investigating impairment of furin substrate processing., Results: Expression of PDX prevented the s.c. growth of the transfected cells. Invasion assays demonstrated that PDX-transfected cells exhibited a reduced invasive ability in vitro and in vivo. Furthermore, s.c. growth of PDX transfectant xenotransplants showed a significant reduction in size that coincided with a significant decrease of the in vitro doubling time and of the in vivo cell proliferation ability. Additional studies showed that the furin substrates insulin-like growth factor IR, transforming growth factor beta and membrane type 1-matrix metalloprotease were not activated in PDX-expressing astrocytoma cells., Conclusions: PDX expression in astrocytoma cells demonstrated a direct mechanistic link between furin inhibition, and decreased astrocytoma proliferation and invasive ability. Because furin inhibition inhibits both invasiveness and cell growth in astrocytoma, furin should be considered a promising target for glioblastoma therapy.