Ricardo Lopez de Cicco

Proprotein convertases: “Master switches” in the regulation of tumor growth and progression

Proprotein convertases (PCs) are a group of Ca2+‐dependent serine proteases that have homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This group is comprised of less than a dozen members, known as furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, PC7/PC8/LPC, SKI/S1P, and NARC‐1/PCSK9. Four PCs (Furin, PACE4, PC5, and PC7) have been localized to several different tissues and epithelial or nervous system tumors. PCs activate their cognate substrates by limited proteolysis at the consensus sequence RXR/KR↓. Many PC substrates are well known cancer‐associated proteins such as growth factors, growth factor receptors, integrins, and matrix metalloproteases (MMPs). For example, IGF‐1 and its receptor, TGF‐β, VEGF‐C, and MT‐MMPs have direct roles in tumor progression and metastasis. Furin, a well‐studied member of the PC family, has been associated with enhanced invasion and proliferation in head and neck, breast, and lung cancer. Conversely, inhibition of PC activity by PDX or several PC pro‐segments, resulted in reduced processing of these key cancer‐related substrates in human squamous cell carcinomas (SCC), colon adenocarcinoma, and astrocytoma cell lines. In parallel to these changes in cell proliferation and invasiveness as well as metastatic ability were markedly impaired. By controlling the maturation/activation of key cancer‐associated proteins, PCs act as “master switches” at different levels during tumor development and progression. The manifold effects of PCs, influencing tumor cell proliferation, motility, adhesiveness, and invasiveness, should be exploited by further developing competitive/inhibitory therapeutic strategies that would be able to neutralize simultaneously the most salient cancer cell properties.

Furin inhibition results in absent or decreased invasiveness and tumorigenicity of human cancer cells

Pro-protein convertases such as furin are expressed in many human tumor lines and primary tumors. Furin processes stromelysin-3, membrane type 1 matrix metalloproteinase (MMPs) involved in tumor cell invasiveness, as well as growth factors such as transforming growth factor β1. Evaluation of furin expression in head and neck squamous cell carcinoma (HNSCC) cells exhibiting different invasive ability showed that furin overexpression correlated with their respective invasiveness. The use of a selective furin inhibitor, alpha 1-PDX (PDX) was studied in three furin-expressing invasive HNSCC cell lines. The effects of PDX transfection were evaluated in vivo and in vitro to determine changes in the malignant phenotype. Transfection of HNSCC cell lines with PDX resulted in significant decrease or absence of tumorigenicity after s.c. inoculation into severe combined immunodeficient mice. Likewise, in vitro invasiveness was reduced ≈50%. The in vivo invasion assay using tracheal xenotransplants showed even more drastic reductions of the invasive ability of PDX-transfected cells (up to an 80% decrease). PDX-transfected cells did not invade or penetrated less into the tracheal wall tissues than their vector alone-transfected counterparts. In addition, the former cells showed a remarkable decrease in MMP-2 processing and activity. After PDX transfection the cells were less efficient in processing the tumor progression-associated furin substrates transforming growth factor β1 and pro-membrane type 1-MMP. These findings indicate that furin inhibition is a feasible approach to attenuate and even abolish certain critical attributes of the advanced malignant phenotype. Thus, furin should be considered as a promising target for cancer therapy.

Elevated furin expression in aggressive human head and neck tumors and tumor cell lines

Pro‐protein convertases (PCs) are proteases that recognize and cleave precursor proteins. Furin, a well‐studied PC, is ubiquitously expressed, and it has been implicated in many physiological and pathological processes. Some substrates for furin, such as membrane type 1 (MT1) matrix metalloproteinase (MMP), an MMP that activates gelatinase, a collagen‐degrading enzyme, are associated with the advanced malignant phenotype. This report examines the expression of furin in carcinoma cell lines of different invasive ability. The levels of furin mRNA and protein correlated with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. Furin expression also was investigated in primary head and neck squamous cell carcinomas (HNSCCs). Furin mRNA was not detected in nonmetastasizing carcinomas. In contrast, furin mRNA was expressed in metastasizing HNSCCs. Immunohistochemistry and Western blot analysis confirmed these results at the protein level. Furin activity was investigated indirectly by evaluating the expression of the pro‐form and the processed form of MT1‐MMP. Metastasizing HNSCCs showed increased expression of MT1‐MMP. Furthermore, pro‐MT1‐MMP expression was noted in most of the nonmetastasizing HNSCCs analyzed by Western blot, and it was absent in the metastasizing HNSCCs. This finding suggests a lower level of furin‐mediated MT1‐MMP activation in the less aggressive cancers. These observations indicate that furin plays a role in tumor progression. Its overexpression in more aggressive or metastasizing cancers resulted in increased MMP processing.

Increased Furin Activity Enhances the Malignant Phenotype of Human Head and Neck Cancer Cells

Many proteins are synthesized as inactive proforms requiring a proteolytic processing to render them active. A variety of proteases catalyze these cleavage reactions. Proprotein convertases are a family of serine proteases capable of activating substrates that will subsequently intervene in extracellular matrix (ECM) degradation, cell growth, differentiation and viral pathogenesis. Furin, the prototype of this family, has been implicated in many physiological and pathological processes. Some of its substrates such as TGF-beta, MT-MMPs, and IGFR-1 have been identified. Overexpression of furin has been observed in several human tumors. In this report we demonstrate that overexpression of furin causes a significant increase in the invasive potential of human tumor cells of low and moderate aggressive potential in vitro and in vivo. SCC12 and SCC15 were transfected with furin cDNA, resulting in efficient processing of furin substrates. An in vivo invasion assay showed enhancement of invasive ability. Inhibition of furin activity with the synthetic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl-ketone, CMK, showed a significant decrease in both processing and in vitro invasiveness. A moderate enhancement in proliferation rate was observed when cells were transfected with furin. CMK treatment resulted in a marked reduction of this effect. Tumors obtained after subcutaneous (s.c.) inoculation of furin-overexpressing cells were larger and developed earlier than the controls. Furin overexpression caused an imbalance in the activation of invasion and proliferation-related substrates leading to the acquisition of an advanced malignant phenotype. In addition, inhibition of furin activity decreases substrate activation, proliferation rate, and invasive potential of cancer cells, suggesting that furin is a potentially useful target for therapeutics.

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.

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 ∼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.

Stromelysin‐1/matrix metalloproteinase‐3 (MMP‐3) expression accounts for invasive properties of human astrocytoma cell lines

Tumor invasiveness is an intrinsic feature of most glial tumors that accounts for their malignant and locally destructive nature. We evaluated the subcutaneous (sc) tumorigenicity and in vivo invasiveness of 9 astrocytoma cell lines together with their respective metalloprotease activity in order to establish their biologic behavior and malignant potential. Invasiveness was assessed with an in vivo invasion assay using tracheal xenotransplants subcutaneously implanted into Scid mice. This assay permitted us to evaluate the penetration of tumor cells into the transplanted deepithelialized tracheas previously inoculated with either normal primary glial cells or with astrocytoma‐derived cell lines. Although only 2 cell lines were tumorigenic after sc inoculation, 5 out of 9 tumor cell lines were tumorigenic in the tracheal graft system. The astrocytoma cell lines showed varying levels of penetration into the tracheal wall. The tumor lines GOS3, M059K, CCFSTTG1 and A172, as well as primary normal astrocytes, were nontumorigenic and noninvasive in this experimental model. LN405, SW1088 and SW1783 cells that were not tumorigenic as sc xenotransplants, on the other hand, grew well in the tracheal graft system showing low levels of in vivo invasiveness. U87MG and U118MG cells were tumorigenic as sc xenotransplants and showed high levels of invasiveness. In parallel to these in vivo studies, the constitutive levels of secreted gelatinases and stromelysins (MMP‐3 and MMP‐11) were investigated using conditioned media submitted to gelatin or casein‐substrate zymography and Western blot analysis. Neither the gelatinases (MMP‐2 and MMP‐9) nor MMP‐11 showed a direct correlation with the levels of in vivo tumor cell invasiveness. Conversely, secretion of MMP‐3 correlated closely with tumorigenicity and invasiveness. In vitro tumor cell invasiveness was significantly reduced after incubation with the metalloproteinase inhibitor GM6001. This positive correlation between MMP‐3 and the depth of tracheal wall penetration led us to conclude that the invasive properties of brain tumor cells may be due to the direct or indirect proteolytic effects of MMP‐3 on extracellular matrix (ECM) macromolecules and that this enzyme might be a potential target for future therapies.

Characterization of a Genomic Signature of Pregnancy Identified in the Breast

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. Cancer Prev Res; 4(9); 1457–64. ©2011 AACR.

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.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation‐associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full‐term pregnancy.