Platelet-derived growth factor D is activated by urokinase plasminogen activator in prostate carcinoma cells

Platelet-derived growth factor (PDGF) protein family members are potent mitogens and chemoattractants for mesenchymal cells. The classic PDGF ligands A and B are single-domain protein chains which are secreted as active dimers capable of activating their cognate PDGF receptors (PDGFRs). In contrast to PDGFs A and B, PDGF D contains an N-terminal complement subcomponent C1r/C1s, Uegf, and Bmp1 (CUB) domain and a C-terminal PDGF domain. PDGF D must undergo extracellular proteolytic processing, separating the CUB domain from the PDGF domain, before the PDGF domain can stimulate beta-PDGFR-mediated cell signal transduction. Here, we report that prostate carcinoma cells LNCaP and PC3 autoactivate latent full-length PDGF D into its active form under serum-independent conditions and that this autoactivation is inhibited by PAI-1, a urokinase plasminogen activator (uPA)/tissue plasminogen activator (tPA) inhibitor. Interestingly, uPA, but not the closely related protease tPA, is capable of processing recombinant latent PDGF DD into the active form. We identify the uPA cleavage site between the CUB and PDGF domains of the full-length PDGF D by mutational analysis and show that PDGF D and uPA colocalize in human prostate carcinoma. This evidence provides a direct link between uPA- and PDGF D-mediated cell signaling, which may contribute to the progression of prostate cancer.     

TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer

TMEFF2 is a type I transmembrane protein with two follistatin (FS) and one EGF‐like domain over‐expressed in prostate cancer; however its biological role in prostate cancer development and progression remains unclear, which may, at least in part, be explained by its proteolytic processing. The extracellular part of TMEFF2 (TMEFF2‐ECD) is cleaved by ADAM17 and the membrane‐retained fragment is further processed by the gamma‐secretase complex. TMEFF2 shedding is increased with cell crowding, a condition associated with the tumour microenvironment, which was mediated by oxidative stress signalling, requiring jun‐kinase (JNK) activation. Moreover, we have identified that TMEFF2 is also a novel substrate for other proteases implicated in prostate cancer, including two ADAMs (ADAM9 and ADAM12) and the type II transmembrane serine proteinases (TTSPs) matriptase‐1 and hepsin. Whereas cleavage by ADAM9 and ADAM12 generates previously identified TMEFF2‐ECD, proteolytic processing by matriptase‐1 and hepsin produced TMEFF2 fragments, composed of TMEFF2‐ECD or FS and/or EGF‐like domains as well as novel membrane retained fragments. Differential TMEFF2 processing from a single transmembrane protein may be a general mechanism to modulate transmembrane protein levels and domains, dependent on the repertoire of ADAMs or TTSPs expressed by the target cell.     

The matriptase-prostasin proteolytic cascade in epithelial development and pathology

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton’s Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.     

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.     

EGF promotes the shedding of soluble E-cadherin in an ADAM10-dependent manner in prostate epithelial cells

During the progression of prostate cancer, the epithelial adhesion molecule E-cadherin is cleaved from the cell surface by ADAM15 proteolytic processing, generating an extracellular 80 kDa fragment referred to as soluble E-cadherin (sE-cad). Contrary to observations in cancer, the generation of sE-cad appears to correlate with ADAM10 activity in benign prostatic epithelium. The ADAM10-specific inhibitor INCB8765 and the ADAM10 prodomain inhibit the generation of sE-cad, as well as downstream signaling and cell proliferation. Addition of EGF or amphiregulin (AREG) to these untransformed cell lines increases the amount of sE-cad shed into the conditioned media, as well as sE-cad bound to EGFR. EGF-associated shedding appears to be mediated by ADAM10 as shRNA knockdown of ADAM10 results in reduced shedding of sE-cad. To examine the physiologic role of sE-cad on benign prostatic epithelium, we treated BPH-1 and large T immortalized prostate epithelial cells (PrEC) with an sE-cad chimera comprised of the human Fc domain of IgG₁, fused to the extracellular domains of E-cadherin (Fc-Ecad). The treatment of untransformed prostate epithelial cells with Fc-Ecad resulted in phosphorylation of EGFR and downstream signaling through ERK and increased cell proliferation. Pre-treating BPH-1 and PrEC cells with cetuximab, a therapeutic monoclonal antibody against EGFR, decreased the ability of Fc-Ecad to induce EGFR phosphorylation, downstream signaling, and proliferation. These data suggest that ADAM10-generated sE-cad may have a role in EGFR signaling independent of traditional EGFR ligands.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acid-sensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two-chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.     

The PDGF system and its antagonists in liver fibrosis

Platelet derived growth factor (PDGF) signaling plays an important role in activated hepatic stellate cells and portal fibroblast proliferation, chemotaxis, migration and cell survival. PDGF receptors and ligands are upregulated in experimental liver fibrotic models as well as in human liver fibrotic diseases. Blocking of PDGF signaling ameliorates experimental liver fibrogenesis. The plurality of molecular and cellular activities of PDGF and its involvement in initiation, progression and resolution of hepatic fibrogenesis offers an infinite number of therapeutic possibilities. These include the application of therapeutic antibodies (e.g. AbyD3263, MOR8457) which specifically sequester individual PDGF isoforms or the inhibition of PDGF isoforms by synthetic aptamers. In particular, the isolation of innovative slow off-rate modified aptamers (e.g., SOMAmer SL1 and SL5) that carry functional groups absent in natural nucleic acids by the Systematic Evolution of Ligands by EXponential (SELEX) enrichment technique offers the possibility to design high affinity aptamers that target PDGF isoforms for clinical purposes. Dominant-negative soluble PDGF receptors are also effective in attenuation of hepatic stellate cell proliferation and hepatic fibrogenesis. Moreover, some multikinase inhibitors targeting PDGF signaling have been intensively tested during the last decade and are on the way into advanced preclinical studies and clinical trials. This narrative review aims to gauge the recent progression of research into PDGF systems and liver fibrosis.     

PDGF signaling from pharyngeal pouches promotes arch artery morphogenesis

The great vessels of the heart originate from the pharyngeal arch arteries(PAAs).Anomalies of the PAAs often occur together with pharyngeal pouch malfo rmations,but the reasons for this phenomenon are not fully understood.In the current study,we show that platelet-derived growth factor(PDGF) signaling derived from the pharyngeal pouches plays an important function in PAA vasculogenesis,During PAA development in zebrafish embryos,pdgfaa and pdgfab are expressed in the developing pharyngeal pouches.Results from loss-of-function experiments revealed a critical role of these genes in PAA formation.We found that nitroreductase(NTR)-mediated pouch ablation distinctly decreased PDGF receptor tyrosine phosphorylation,yielding a severe loss of PAAs.Importantly,pouch-specific overexpression of pdgfaa in pdgfaa~(-/-);pdgfab~(-/-)mutants significantly relieved the PAA defects,which indicated a primary role of pharyngeal pouch-expressed PDGF ligands in signal activation and PAA morphogenesis.Our findings further showed that PDGF signaling was indispensable for the proliferation of PAA angioblasts.Together,these results established a role for PDGFaa-and PDGFab-mediated tissuetissue interaction during PAA development.     

Cyclin D1 Induction by Benzo[a]pyrene-7,8-diol-9,10-epoxide via the Phosphatidylinositol 3-Kinase/Akt/MAPK- and p70s6k-dependent Pathway Promotes Cell Transformation and Tumorigenesis

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of B[a]P, has been well recognized as one ubiquitous carcinogen, but the molecular mechanism involved in its carcinogenic effect remains obscure. In the present study, we found that bronchial epithelial cells (Beas-2B) and hepatocytes treated with B[a]PDE presented a significant increase of cyclin D1 expression. Moreover, Akt, p70^s6k, and MAPKs including JNK, Erks, and p38 were notably activated in B[a]PDE-treated Beas-2B cells, whereas NF-κB, NFAT, and Egr-1 were not. Our results demonstrated that JNK and Erks were required in B[a]PDE-induced cyclin D1 expression because the inhibition of JNK or Erks by a selective chemical inhibitor or dominant negative mutant robustly impaired the cyclin D1 induction by B[a]PDE. Furthermore, we found that overexpression of the dominant negative mutant of p85 (regulatory subunit of phosphatidylinositol 3-kinase) or Akt dramatically suppressed B[a]PDE-induced JNK and Erk activation as well as cyclin D1 expression, suggesting that cyclin D1 induction by B[a]PDE is via the phosphatidylinositol 3-kinase/Akt/MAPK-dependent pathway. In addition, we clarified that p70^s6k is also involved in B[a]PDE-induced cyclin D1 expression because rampamycin pretreatment dramatically reduced cyclin D1 induction by B[a]PDE. More importantly, we demonstrated that up-regulated cyclin D1 by B[a]PDE plays a critical role in oncogenic transformation and tumorigenesis of Beas-2B cells. These results not only broaden our knowledge of the molecular mechanism of B[a]PDE carcinogenicity but also lead to the further study of chemoprevention of B[a]PDE-associated human cancers.     

Targeting the PDGF signaling pathway in tumor treatment

Platelet-derived growth factor (PDGF) isoforms and PDGF receptors have important functions in the regulation of growth and survival of certain cell types during embryonal development and e.g. tissue repair in the adult. Overactivity of PDGF receptor signaling, by overexpression or mutational events, may drive tumor cell growth. In addition, pericytes of the vasculature and fibroblasts and myofibroblasts of the stroma of solid tumors express PDGF receptors, and PDGF stimulation of such cells promotes tumorigenesis. Inhibition of PDGF receptor signaling has proven to useful for the treatment of patients with certain rare tumors. Whether treatment with PDGF/PDGF receptor antagonists will be beneficial for more common malignancies is the subject for ongoing studies.     

The epidermal growth factor receptor (EGFR) is proteolytically modified by the Matriptase-Prostasin serine protease cascade in cultured epithelial cells

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.     

Calcitriol inhibits growth response to Platelet-Derived Growth Factor-BB in human prostate cells

Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRalpha and beta proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRbeta and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRbeta expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRalpha and PDGF-B mRNA expression. We suggest that inhibition of PDGFRbeta expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB.     

Strong expression association between matriptase and its substrate prostasin in breast cancer

Breast cancer tumorigenesis is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and signaling receptors that are critically involved in neoplastic progression. Multiple studies implicate the membrane anchored serine protease matriptase (also known as MT-SP1 and epithin) in breast cancer. The pro-form of the GPI-anchored serine protease prostasin has recently been identified as a physiological substrate of matriptase and the two proteases are co-expressed in multiple healthy tissues. In this study, the inter-relationship between the two membrane-anchored serine proteases in breast cancer was investigated using breast cancer cell lines and breast cancer patient samples to delineate the association between matriptase and prostasin. We used Western blotting to determine the expression of matriptase and prostasin proteins in a panel of breast cancer cell lines and immunohistochemistry to assess the expression in serial sections from breast cancer tissue arrays. We demonstrate that the expression of matriptase and prostasin is closely correlated in breast cancer cell lines as well as in breast cancer tissue samples. Furthermore, matriptase and prostasin display a near identical spatial expression pattern in the epithelial compartment of breast cancer tissue. These data suggest that the matriptase-prostasin cascade might play a critical role in breast cancer.     

Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule.

We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.     

EGF receptor signaling in prostate morphogenesis and tumorigenesis.

The growth and differentiation of the prostate gland are largely dependent on extracellular signaling factors. In addition to androgens, many polypeptide growth factors function through autocrine or paracrine networks. The paracrine interaction between stromal and epithelial cells is critical for androgen regulation, morphogenesis, epithelial cell proliferation, and secretory differentiation. Efforts to identify the essential growth factors and studies on their effects have been prompted by the fact that prostate cells in culture need substances other than androgens for proliferation. In this context, transforming growth factor-alpha and epidermal growth factor, among others, have been studied extensively. Recent advances have suggested that these EGF receptor (EGFR) ligands play roles not only during glandular development but also during neoplastic transformation and tumor progression. The cell responses most relevant to the role of this receptor signaling are both mitogenesis and cell motility. The aim of the review is to provide an overview of current knowledge about EGFR and its ligands in the organogenesis and tumorigenesis of the prostate gland.     

Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting

Background

Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.

Methods and Findings

We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.

Conclusions

This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.     

Bovine type I collagen inhibits Raw264.7 cell proliferation through phosphoinositide 3-kinase- and mitogen-activated protein kinase-dependent down-regulation of cyclins D1, A and B1

Bovine type I collagen (BIC), which is widely used as a fibrous extracellular matrix component in cell culture models, inhibits the progression of melanoma cell cycle via p27 up-regulation. BIC also induces nitric oxide synthase in macrophages through JunB/AP-1 and NF-kappaB activation. Given the previous observations, this study investigates the effect of BIC on the cell cycle progression and regulatory function of Raw264.7 macrophage cells and the responsible signaling pathways. Cell cycle analysis revealed that BIC completely suppressed proliferation of Raw264.7 cells with inhibition of the percentage of cells in the S phase and the reciprocal decrease in the G0/G1 phase. DNA synthesis was also inhibited by BIC, as evidenced by a decrease in the cellular incorporation of [3H]thymidine. The G1/S arrest induced by BIC was reversed by chemical inhibition of phosphatidylinositol 3-kinase (PI3-kinase) or overexpression of the p85 subunit of PI3-kinase. Either PD98059 or stable transfection with mitogen-activated protein kinase kinase-1 [MKK1(-)] or c-Jun N-terminal kinase 1 [JNK1(-)] also released the cell cycle arrest. Immunoblot analyses revealed that the levels of cyclins D1, A and B1 were partly or completely down-regulated by BIC, but cyclin E, p21 and p27 were minimally changed. Chemical inhibition and dominant negative mutant overexpression experiments revealed that either PI3-kinase inhibition or JNK1(-) transfection prevented the decreases in cyclin D1, A and B1 by BIC, indicating that the PI3-kinase and JNK1 pathways were associated with disruption of the cyclins. The pathway involving MKK1-extracellular signal-regulated kinase-1/2 (ERK1/2) was responsible for the suppression of cyclin A and B1, but not that of cyclin D1. The present study showed that BIC inhibited proliferation of Raw264.7 cells and that the pathways involving PI3-kinase and mitogen-activated protein kinases regulate the cell cycle arrest.     

Novel PDGF family members: PDGF-C and PDGF-D

Platelet-derived growth factors (PDGFs) were discovered almost two decades ago. The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well studied and they regulate a number of physiological and pathophysiological processes in many types of mesenchymal cells via two receptor tyrosine kinases, PDGF receptors alpha and beta. Recently, two additional PDGF polypeptide chains were discovered, namely PDGF-C and PDGF-D. The discovery of two additional ligands for the two PDGF receptors suggests that PDGF-mediated signaling is more complex than previously anticipated.