Targeting proprotein convertases in furin-rich lung cancer cells results in decreased in vitro and in vivo growth.

Proprotein convertases (PCs) are serine proteases with an active role in the post-translational processing of numerous inactive proteins to active proteins including many substrates of paramount importance in cancer development and progression. Furin (PCSKC3), a well-studied member of this family, is overexpressed in numerous human and experimental malignancies. In the present communication, we treated two furin-overexpressing non-small cell carcinoma (NSCLC) cell lines (Calu-6 and HOP-62) with the PC inhibitor CMK (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone). This resulted in a diminished IGF-1R processing and a simultaneous decrease in cell proliferation of two NSCLC lines. Similarly, growth of subcutaneous xenografts of both cell lines, were partially inhibited by an in vivo treatment with the same drug. These observations point to a potential role of PC inhibitors in cancer therapy. © 2016 Wiley Periodicals, Inc.

Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6).

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

Physico-mechanical aspects of extracellular matrix influences on tumorigenic behaviors.

Tumor progression in vitro has traditionally been studied in the context of two-dimensional (2D) environments. However, it is now well accepted that 2D substrates are unnaturally rigid compared to the physiological substrate known as extracellular matrix (ECM) that is in direct contact with both normal and tumorigenic cells in vivo. Hence, the patterns of interactions, as well as the strategies used by cells in order to penetrate the ECM, and migrate through a three-dimensional (3D) environment are notoriously different than those observed in 2D. Several substrates, such as collagen I, laminin, or complex mixtures of ECM components have been used as surrogates of native 3D ECM to more accurately study cancer cell behaviors. In addition, 3D matrices developed from normal or tumor-associated fibroblasts have been produced to recapitulate the mesenchymal 3D environment that assorted cells encounter in vivo. Some of these substrates are being used to evaluate physico-mechanical effects on tumor cell behavior. Physiological 3D ECMs exhibit a wide range of rigidities amongst different tissues while the degree of stromal stiffness is known to change during tumorigenesis. In this review we describe some of the physico-mechanical characteristics of tumor-associated ECMs believed to play important roles in regulating epithelial tumorigenic behaviors.

Keep recycling going: New approaches to reduce LDL-C.

Hypercholesterolemia represents a leading cause in the development of atherosclerotic plaques, increasing the risk for ACVS. It actually counts as a major cause of cardiovascular disease etiopathogenesis. The causes of hypercholesterolemia are multifactorial, spanning from genetic constitution, age, sex, to sedentary lifestyle and diets rich in sugars and lipids. Although dietary restriction in saturated fats, increased exercise, and other modification in lifestyle represent a first-line approach to treat very initial stages in hypercholesterolemia, most patients will require the addition of pharmacological agents. Pharmacological approaches include inhibition of cholesterol synthesis, decreased fat absorption from the GI tract, and increased degradation of FA. These strategies present a series of side effects, low therapeutic efficiency in some patients, and reduced tolerability. One of the major goals in treatment for hypercholesterolemia is to decrease the levels of low density lipoproteins (LDL), while maintaining those of high density lipoproteins (HDL). LDL particles contain about 80% of lipids, most of it cholesterol and cholesteryl esters, and 20% of the ApoB-100 protein. LDL carries cholesterol to the tissues, to be incorporated to biological membranes, or to be transformed to steroids. Excess of LDL translates into increased levels of circulating cholesterol particles and accumulation in certain tissues, especially vascular tissue, initiating a fatty streak, which may evolve to an atheroma, causing a series of cardiovascular problems, including impaired circulation, high blood pressure, increased cardiac workload, and coronary artery disease. It is essential to prevent LDL accumulation into the bloodstream to avoid the formation of these fatty streaks and the initiation of a cascade that will lead to the development of atherosclerosis. In healthy individuals. Under physiological conditions, LDL is effectively removed from circulation through receptor-mediated endocytosis. LDL clearance involves binding to its receptor, LDLR, which enables the internalization of the LDL particle and drives its degradation in lysosomes. Once the LDL particle is degraded, the free receptor recycles to the plasma membrane, and captures new LDL particles. Adequate levels of LDLR are essential to remove the excess of cholesterol-laden LDL. Proprotein convertase, subtilysin kexin type 9 (PCSK-9), expressed in liver and intestine, binds to LDLR, and internalized. Once inside the cell, PCSK-9 catalyzes the proteolysis of LDLR, preventing its recycling to the cell surface, and effectively decreasing the number of LDLR, notoriously decreasing the ability to clear LDL from circulation. Levels of PCSK-9 varies with age, gender, and levels of insulin, glucose, and triglycerides. Loss-of-function mutations in PCSK-9 gene invariably translates into lower levels of LDL, and decreased risk of developing coronary artery disease. Conversely, increased activity or expression of this enzyme leads to hypercholesterolemia. Inhibition of PCSK9 has proven to be successful in decreasing LDL levels and risk of the development of hypercholesterolemia with its associated higher risk for ASCVD. Patient with gain-of-function mutations in the PCSK9 undoubtedly benefit from therapies based on PCSK-9 inhibitors. However, millions of patients show statin intolerance, or cannot be efficiently controlled by statins alone- the most prevalent therapy for hypeprcholesterolemia. This commentary will evaluate the possibilities, caveats and future directions in the treatment of hypercholesterolemia, and therapies with combination of drugs.

Increased expression of the pro-protein convertase furin predicts decreased survival in ovarian cancer.

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.

PACE4 expression in mouse basal keratinocytes results in basement membrane disruption and acceleration of tumor progression.

Collagen type IV degradation results in disruption and breakdown of the normal basement membrane architecture, a key process in the initiation of tumor microinvasion into the connective tissue. PACE4, a proprotein convertase, activates membrane type matrix metalloproteinases (MT-MMPs) that in turn process collagenase type IV. Because PACE4 is overexpressed in skin carcinomas and in vitro overexpression of PACE4 resulted in enhanced invasiveness, we investigated whether or not in vivo PACE4 expression leads to the acquisition of invasiveness and increased tumorigenesis. Two transgenic mouse lines were designed by targeting PACE4 to the epidermal basal keratinocytes. Transgenic keratinocytes showed increased processing of MT1-MMP and MT2-MMP resulting in collagenase IV activation and collagen type IV degradation. Higher collagenolytic activity partially disrupted normal basement membrane architecture favoring epithelial endophytic growth into the dermis and accelerating invasion and metastasis after chemical carcinogenesis. PACE4 overexpression resulted in enhanced susceptibility to carcinogenesis and tumor progression pointing to a new target for blocking tumor cell invasiveness.

Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.

Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-beta, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies.

Proprotein convertase inhibition: Paralyzing the cell's master switches.

Proprotein convertases are serine proteases responsible for the cleavage and subsequent activation of protein substrates, many of them relevant for the development of an ample variety of diseases. Seven of the PCs, including furin and PACE4, recognize and hydrolyze the C-terminal end of the general sequence RXRR/KXR, whereas PCSK-9 recognizes a series of non-basic amino acids. In some systems, PC-mediated substrate activation results in the development of pathological processes, such as cancer, endocrinopathies, and cardiovascular and infectious diseases. After establishing PCs as relevant contributors to disease processes, research efforts were directed towards the development of inhibition strategies, including small and large molecules, anti-sense therapies, and antibody-based therapies. Most of these inhibitors mimic the consensus sequence of PCs, blocking the active site in a competitive manner. The most promising inhibitors were designed as bioengineered proteins; however, some non-protein and peptidomimetic agents have also proved to be effective. These efforts led to the design of pre-clinical studies and clinical trials utilizing inhibitors to PCs. Although the initial studies were performed using non-selective PCs inhibitors, such as CMK, the search for more specific, and compartmentalized selective inhibitors resulted in specific activities ascribed to some, but not all of the PCs. For instance, PACE4 inhibitors were effective in decreasing prostate cancer cell proliferation, and neovascularization. Decreased metastatic ovarian cancer utilizing furin inhibitors represents one of the major endeavors, currently in a phase II trial stage. Antibodies targeting PCSK-9 decreased significantly the levels of HDL-cholesterol, in a phase III trial. The study of Proprotein convertases has reached a stage of maturity. New strategies based on the alteration of their activity at the cellular and clinical level represent a promising experimental pharmacology field. The development of allosteric inhibitors, or specific agents directed against individual PCs is one of the challenges to be unraveled in the future.

Overexpression of the calcium sensor visinin-like protein-1 leads to a cAMP-mediated decrease of in vivo and in vitro growth and invasiveness of squamous cell carcinoma cells.

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.

Simultaneous expression of furin and vascular endothelial growth factor in human oral tongue squamous cell carcinoma progression.

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.

Inhibition of furin-mediated processing results in suppression of astrocytoma cell growth and invasiveness.

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.

Biosynthesis of a substituted cellulose from a mutant strain of Xanthomonas campestris.

In Xanthomonas campestris the genes involved in polysaccharide (xanthan) biosynthesis are located in a gene cluster (gum) of 16 kb. A Tn5 insertion mutant with a reduced slimy phenotype has been characterized. This mutant failed to produce the pentasaccharide repeating-unit of xanthan. Only three sugars were transferred to the prenyl phosphate intermediate. Several lines of evidence suggested that the lipid-associated saccharide was the trisaccharide reducing end of the pentasaccharide from the wild-type strain. This trisaccharide was built up from UDP-Glc and GDP-Man, and a glucose residue was at the reducing end, linked to an allylic prenol through a diphosphate bridge. Results from one- or two-stage reactions showed that the trisaccharide-P-P-polyprenol was the precursor of the polymer. This new polymer, a polytrisaccharide, was detected also in vivo. The transposon responsible for the mutation was located within gumK gene. Therefore, this gene encodes for the glycosyltransferase IV, which catalyses the transfer of glucuronic acid to the lipid-linked beta-D-Manp-(1-->3)-beta-D-Glcp-(1-->4)-beta-D-Glcp trisaccharide. A recombinant plasmid with the whole gum cluster restored the wild type phenotype.

Enhanced UV-induced skin carcinogenesis in transgenic mice overexpressing proprotein convertases.

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.

The mesenchymal tumor microenvironment: a drug-resistant niche.

Drug and radiation resistance represent a challenge for most anticancer therapies. Diverse experimental approaches have provided evidence that the tumor-associated microenvironment constitutes both a protective shell that impedes drug or radiation access and a permissive or promotive microenvironment that encourages a nurturing cancer (i.e., cancer stem cell) niche where tumor cells overcome treatment- and cancer-induced stresses. Better understanding of the effects of the tumor microenvironment on cancer cells before, during and immediately after chemo- or radiotherapy is imperative to design new therapies aimed at targeting this tumor-protective niche. This review summarizes some of the known mesenchymal stromal effects that account for drug resistance, the main signal transduction pathways associated with this resistance and the therapeutic efforts directed to increase the success of current therapies. Special emphasis is given to environment-mediated drug resistance in general and to cell adhesion-mediated drug resistance in particular.

Transgenic overexpression of the proprotein convertase furin enhances skin tumor growth.

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 ß, 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.

Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma.

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.

Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

VILIP-1 expression in vivo results in decreased mouse skin keratinocyte proliferation and tumor development.

VILIP-1, a member of the neuronal Ca(2+) 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 approximately 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.