Daniel E. Bassi

The DNA repair gene MBD4 (MED1) is mutated in human carcinomas with microsatellite instability.

The DNA repair gene MBD4 ( MED1 ) is mutated in human carcinomas with microsatellite instability

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.

Stroma-Derived Three-Dimensional Matrices Are Necessary and Sufficient to Promote Desmoplastic Differentiation of Normal Fibroblasts

Stromagenesis is a host reaction of connective tissue that, when induced in cancer, produces a progressive and permissive mesenchymal microenvironment, thereby supporting tumor progression. The stromal microenvironment is complex and comprises several cell types, including fibroblasts, the primary producers of the noncellular scaffolds known as extracellular matrices. The events that support tumor progression during stromagenesis are for the most part unknown due to the lack of suitable, physiologically relevant, experimental model systems. In this report, we introduce a novel in vivo-like three-dimensional system derived from tumor-associated fibroblasts at diverse stages of tumor development that mimic the stromagenic features of fibroblasts and their matrices observed in vivo. Harvested primary stromal fibroblasts, obtained from different stages of tumor development, did not retain in vivo stromagenic characteristics when cultured on traditional two-dimensional substrates. However, they were capable of effectively maintaining the tumor-associated stromal characteristics within three-dimensional cultures. In this study, we demonstrate that in vivo-like three-dimensional matrices appear to have the necessary topographical and molecular information sufficient to induce desmoplastic stroma differentiation of normal fibroblasts.

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.

The Proprotein Convertases Furin and PACE4 Play a Significant Role in Tumor Progression

Processing of latent precursor proteins by proprotein convertases (PCs) into their biologically active products is a common mechanism required for many important biologic functions. This process is tightly regulated, leading to the generation of active peptides and proteins including neuropeptides and polypeptide hormones, protein tyrosine phosphatases, growth factors and their receptors, and enzymes including matrix metalloproteases (MMPs). These processing reactions occurs at pairs of basic amino acids. Within the past several years, a novel family of Ca2+‐dependent serine proteases has been identified, all of which possess homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This family of PCs is currently comprised of fewer than a dozen members, known as furin/paired basic amino‐acid–cleaving enzyme (PACE), PC1/PC3, PC2, PC4, PACE4, PC5/PC6, and PC7/PC8/lymphoma proprotein convertase. They share a high degree of amino‐acid identity of 50–75% within their catalytic domains. Despite the relatively high degree of homology in the PC family, only PACE4 and furin localize to the same chromosome: mouse chromosome 7 and human chromosome 15. Recent reports have supported a possible functional role for PCs in tumorigenesis. For instance, convertases have been shown to be expressed in various tumor lines and human primary tumors. Furin and PACE4 process stromelysin 3 (MMP‐11 or Str‐3), an MMP involved in tumor invasion, into its mature, active form. Similarly, a growing family of MMPs, known as membrane‐type metalloproteinases (MT‐MMPs), and growth factors and adhesion molecules such as E‐cadherin show similar amino‐acid motifs and thus could be activated by furin and PACE4. These data, taken together with the high expression levels of PACE4 in 50% of murine chemically induced spindle cell tumors, confer to PACE4 and possibly other PCs a possible functional role in the activation of MMPs and consequently in tumor cell invasion and tumor progression. This was further supported by the remarkable enhancement in the invasive ability of the PACE4‐transfected murine tumor cell lines. Mol. Carcinog. 28:63–69, 2000.

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.

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.

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.