EGF/EGFR upregulates and cooperates with Netrin-4 to protect glioblastoma cells from DNA damage-induced senescence.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. METHODS: Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. RESULTS: Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. CONCLUSIONS: This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness.

BACKGROUND: Glioblastoma is an untreatable brain cancer. The tumors contain a population of stem-like cells which are highly invasive and resistant to therapies. These cells are the main reason for the lethality of glioblastoma. Extracellular guidance molecule netrin-1 promotes the invasiveness and survival of various cancer cell types. We have previously found that netrin-1 activates Notch signaling, and Notch signaling associates with cell stemness. Therefore, we have here investigated the effects of netrin-1 on glioblastoma pathogenesis and glioblastoma cell stemness. METHODS: Glioma tissue microarrays were stained with immunohistochemistry and the results were used to evaluate the association between netrin-1 and survival of glioma patients. The localization of netrin-1 was analyzed utilizing fresh frozen glioblastoma tissues. The glioma cell invasion was investigated using ex vivo glioma tissue cultures and newly established primary cell cultures in 3D in vitro invasion assays. Intracranial mouse xenograft models were utilized to investigate the effects of netrin-1 on glioblastoma growth and invasion in vivo. RESULTS: Netrin-1 expression associated with poor patient prognosis in grade II-III gliomas. In addition, its expression correlated with the stem-like cell marker nestin. Netrin-1 overexpression in cultured cells led to increased formation of stem-like cell spheroids. In glioblastoma tumor biopsies netrin-1 localized to hypoxic tumor areas known to be rich in the stem-like cells. In xenograft mouse models netrin-1 expression altered the phenotype of non-invasive glioblastoma cells into diffusively invading and increased the expression of glioma stem-like cell markers. Furthermore, a distinct invasion pattern where netrin-1 positive cells were following the invasive stem-like cells was detected both in mouse models and ex vivo human glioblastoma tissue cultures. Inhibition of netrin-1 signaling targeted especially the stem-like cells and inhibited their infiltrative growth. CONCLUSIONS: Our findings describe netrin-1 as an important regulator of glioblastoma cell stemness and motility. Netrin-1 activates Notch signaling in glioblastoma cells resulting in subsequent gain of stemness and enhanced invasiveness of these cells. Moreover, inhibition of netrin-1 signaling may offer a way to target stem-like cells.

Netrin-1: A regulator of cancer cell motility?

Netrins form a family of secreted and membrane-associated proteins, netrin-1 being the prototype and most investigated member of the family. The major physiological functions of netrin-1 lie in the regulation of axonal development as well as morphogenesis of different branched organs, by promoting the polarity of migratory/invasive front of the cell. On the other hand, netrin-1 acts as a factor preventing cell apoptosis. These events are mediated via a range of different receptors, including UNC5 and DCC-families. Cancer cells often employ developmental pathways to gain survival and motility advantage. Within recent years, there has been increasing number of observations of upregulation of netrin-1 expression in different forms of cancer, and the increased expression of netrin-1 has been linked to its functions as a survival and invasion promoting factor. We review here recent advances in the netrin-1 related developmental processes that may be of special interest in tumor biology, in addition to the known functions of netrin-1 in tumor biology with special focus on cancer cell migration.

MMP16 Mediates a Proteolytic Switch to Promote Cell-Cell Adhesion, Collagen Alignment, and Lymphatic Invasion in Melanoma.

Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis.

Increased intravitreal angiopoietin-2 levels associated with rhegmatogenous retinal detachment.

PURPOSE: To explore factors related to pathogenesis of rhegmatogenous retinal detachment (RRD) and development of proliferative vitreoretinopathy (PVR), vitreous levels of angiopoietin-1 and -2 (Ang-1 and -2), previously undefined in RRD, transforming growth factor-(TGF) ß1, vascular endothelial growth factor (VEGF), erythropoietin (EPO) and proteolytic mediators of extracellular matrix remodelling (MMP-2 and -9) were compared in eyes with RRD and eyes with idiopathic macular hole or pucker. METHODS: Vitreous samples were collected from 117 eyes with RRD (study group) and 40 eyes with macular hole or pucker (control group). Growth factors were measured by ELISA and matrix metalloproteinases (MMPs) by gelatin zymography. RESULTS: The mean vitreous concentrations of Ang-2, MMP-2, and MMP-9 were higher (all p < 0.01), whereas concentration of VEGF was lower (p = 0.01) in eyes with RRD relative to controls. Logistic regression analysis identified Ang-2 concentration as a novel marker of RRD (p = 0.0001, OR 48.7). Ang-1, EPO, and total TGF-ß1 levels were not significantly different between the groups. However, TGF-ß1 and MMP-2 were increased in eyes with total RRD compared to those with local RRD (p ≤ 0.05). In eyes with PVR, no differences were observed in any studied marker as compared with non-PVR eyes. CONCLUSIONS: Current results reveal Ang-2 as a key factor upregulated in RRD. It may co-operate with fibrosis-associated factors and contribute to vascular complications such as breakdown of blood-eye barrier and PVR development.

NETRIN-4 protects glioblastoma cells FROM temozolomide induced senescence.

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.

Ephrin-As, Eph receptors and integrin α3 interact and colocalise at membrane protrusions of U251MG glioblastoma cells.

Glioblastoma is the most common brain cancer. Ephrins and their Eph receptors play important roles in the development of central nervous system and the regulation of cancer cell migration and invasion. In a search for the Eph receptor complexes, we used tandem affinity purification based interaction screening with tagged ephrins A1, A3 and A4 combined with protein identification by mass-spectrometry in U251MG glioblastoma cells. Ephrins bound to Eph receptors, mainly to EphA2 in these cells. Integrin α3 was identified in protein complexes with ephrin-As. Soluble ephrin-A1 colocalised with integrin α3 at the cell surface, and was rapidly endocytosed by the cells. However, integrin α3 did not colocalise with internalised ephrin-A1, whereas EphA2 receptor did. In U251MG cells, integrin α3 colocalised with EphA2 receptor at the cell edges and protrusions. Sites of EphA2-integrin α3 colocalisation were positive for vinculin, focal adhesion kinase and phosphotyrosine, that is, markers for cell adhesion and active signalling. The interaction between ephrin-As, Eph receptors and integrin α3 is plausibly important for the crosstalk between Eph and integrin signalling pathways at the membrane protrusions and in the migration of brain cancer cells.

Netrin-1-induced activation of Notch signaling mediates glioblastoma cell invasion.

Glioblastoma multiforme is an aggressively invasive human brain cancer, which lacks effective treatment. The axonal guidance protein, netrin-1, is overexpressed in glioblastoma tumor biopsies. In Matrigel invasion assays we observed that experimental overexpression of netrin-1 increased cell invasiveness and its downregulation decreased invasiveness. Using tandem affinity purification and mass spectrometry protein identification we found that netrin-1 forms a complex with both Notch2 and Jagged1. Recombinant netrin-1 colocalized with Jagged1 and Notch2 at the cell surface and was also present in the intracellular vesicles with Jagged1, but not with Notch2. Netrin-1 activated Notch signaling and subsequent glioblastoma cell invasion. Interestingly, the recombinant central domain of netrin-1 counteracted the effects of the full-length netrin-1: it inhibited glioblastoma cell invasion and Notch activation by retaining the Notch signaling complex at the cell surface. This finding may give rise to therapeutic applications. These results reveal a new mechanism leading to glioblastoma cell invasion, in which netrin-1 activates Notch signaling.

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFß1 in diabetic eyes undergoing vitrectomy.

PURPOSE: Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-ß1 (totalTGFß1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. METHODS: Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFß1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. RESULTS: Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFß1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). CONCLUSION: The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFß1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.

LTBP-2 confers pleiotropic suppression and promotes dormancy in a growth factor permissive microenvironment in nasopharyngeal carcinoma.

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-ß signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.

Netrin-4 promotes glioblastoma cell proliferation through integrin ß4 signaling.

Netrin-4 is a laminin-related secreted molecule originally found to have roles in neuronal axon migration. Recent studies have indicated that netrin-4 also participates in the development of nonneural tissues and modulates tumor cell proliferation and tumor metastasis. Here we have explored the functions and molecular mechanisms of netrin-4 in glioblastoma multiforme. The suppression of netrin-4 expression in glioblastoma cell lines significantly reduced cell proliferation and motility and increased serum deprivation-induced apoptosis. Using tandem affinity purification combined with protein identification by mass spectrometry, we found that integrin ß(4) interacts with netrin-4 and that it mediates mitogenic effects as well as AKT and mammalian target of rapamycin phosphorylation induced by netrin-4. Interestingly, netrin-4 acted as an inhibitor of cell proliferation in integrin ß(4)-silenced glioblastoma cells, and high concentrations of netrin-4 reduced cell proliferation. The negative effects of netrin-4 on proliferation were mediated by UNC5B. Analysis of more than 400 primary tumors from The Cancer Genome Atlas repository revealed that the expression of netrin-4 is significantly downregulated in glioblastoma and that the reduced expression is linked to poor patient survival time. The expression of integrin ß(4) is increased in glioblastoma, and it predicts poor patient survival time. Current results illustrate a novel mechanism for glioma progression, where glioma cells reduce netrin-4 expression to decrease its inhibitory effects. In parallel, the expression of integrin ß(4) is upregulated to sensitize the cells to low concentrations of netrin-4 for maintaining cell proliferation.

Regulation of TGF-ß storage and activation in the human idiopathic pulmonary fibrosis lung.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-ß (TGF-ß) activation. Although TGF-ß activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-ß storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-ß. Latent TGF-ß-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-ß signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-ß signaling activity, respectively, suggesting that LTBP-1-mediated TGF-ß activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-ß-activating integrin ß8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-ß storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-ß availability and activation in different pulmonary compartments in the fibrotic lung.

Membrane-type-3 matrix metalloproteinase (MT3-MMP) functions as a matrix composition-dependent effector of melanoma cell invasion.

In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

The ECM protein LTBP-2 is a suppressor of esophageal squamous cell carcinoma tumor formation but higher tumor expression associates with poor patient outcome.

Our previous studies of chromosome 14 transfer into tumorigenic esophageal squamous cell carcinoma (ESCC) cell line, SLMT, suggested the existence of tumor suppressor genes on chromosome 14. Gene expression profiling of microcell hybrids and the tumor segregants identified an interesting gene, LTBP-2 (latent transforming growth factor ß binding protein 2), which has been analyzed here for its role in ESCC. LTBP-2 maps to 14q24 and encodes a secreted protein, which is a component of the extracellular matrix microfibrils. LTBP-2 expression was downregulated in ESCC cell lines and tumor tissues. Promoter hypermethylation was found to be involved in LTBP-2 inactivation. Functional studies indicated its tumor-suppressive roles in ESCC. In the in vitro colony formation and Matrigel three-dimensional culture assays, LTBP-2 decreased the colony-forming abilities of ESCC cell lines. LTBP-2 expression was associated with reduction of cell migrating and invasive abilities. LTBP-2 could also reduce the tube-forming ability of endothelial cells. Moreover, LTBP-2 induced tumor suppression in in vivo nude mouse assays. Tissue microarray immunohistochemical staining analysis indicated that LTBP-2 expression is reduced in tumor tissues when compared to normal tissues, and LTBP-2 expression correlated significantly with the survival of ESCC patients. Thus, LTBP-2 appears to play an important role in ESCC.

Latent TGF-ß binding proteins (LTBPs) 1 and 3 differentially regulate transforming growth factor-ß activity in malignant mesothelioma.

Malignant mesothelioma is an aggressive cancer of the pleura with poor prognosis. There is a need to identify new biomarkers and therapeutic targets for this invasive and fatal disease. Transforming growth factor ß (TGF-ß) can promote mesothelioma tumorigenesis through multiple mechanisms. Latent TGF-ß binding proteins (LTBPs) regulate TGF-ß activation by targeting the growth factor into the extracellular matrix from where it can be released and activated. We investigated here the expression patterns of different LTBP isoforms in malignant mesothelioma tissues and in 2 established malignant mesothelioma cell lines. All LTBPs were expressed, but LTBP-3 was the main isoform in healthy pleura and in cultured nonmalignant mesothelial cells. We observed down-regulation of LTBP-3 expression in malignant mesothelioma, which was associated with high P-Smad2 levels indicative of TGF-ß activity specifically in the tumor tissue. Small interfering RNA-mediated suppression of LTBP-3 expression in mesothelioma cells increased the secretion of TGF-ß activity. Immunoreactivity of LTBP-1, on the other hand, was markedly strong in the tumor stroma, which showed significantly lower levels of P-Smad2. A strong negative correlation between LTBP-1 and P-Smad2 immunoreactivity was found, implying that LTBP-1 is not likely to contribute directly to the increased levels of TGF-ß activity in malignant mesothelioma. Current results suggest that LTBPs 1 and 3 may have specific roles in malignant mesothelioma pathogenesis through the regulation of TGF-ß activation in the tumor tissue and the structure of the tumor stroma.

Fibroblast growth factor receptor 4 regulates tumor invasion by coupling fibroblast growth factor signaling to extracellular matrix degradation.

Aberrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to tumor progression and anticancer drug resistance. We describe here a novel mechanism of tumor progression by matrix degradation involving epithelial-to-mesenchymal transition in response to membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas, where they were partially coexpressed at the tumor/stroma border and tumor invasion front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMP-dependent collagen invasion. In this experimental model, knockdown of FGFR4-R388 or MT1-MMP by RNA interference blocked tumor cell invasion and growth in collagen. This was coupled with impaired phosphorylation of FGFR substrate 2 and Src, upregulation of E-cadherin, and suppression of cadherin-11 and N-cadherin. These in vitro results were substantiated by reduced MT1-MMP content and in vivo growth of prostate carcinoma cells after the FGFR4-R388 gene silencing. In contrast, knockdown of the alternative FGFR4-G388 allele enhanced MT1-MMP and invasive tumor cell growth in vivo and within three-dimensional collagen. These results will help to explain the reported association of the FGFR4-R388 variant with the progression and poor prognosis of certain types of tumors.

FGF receptor-4 (FGFR4) polymorphism acts as an activity switch of a membrane type 1 matrix metalloproteinase-FGFR4 complex.

Tumor cells use membrane type 1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the signaling mechanisms that underlie MT1-MMP regulation in cancer have remained unclear. Using a systematic gain-of-function kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP activity in tumor cells. In particular, we discovered an MT1-MMP/FGF receptor-4 (FGFR4) membrane complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progression-associated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal stabilization, and surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388 internalization and dissociation of cell-cell junctions. In contrast, the alternative FGFR4-G388 variant down-regulated MT1-MMP, and the overexpression of MT1-MMP and particularly its phosphorylation-defective mutant vice versa induced FGFR4-G388 degradation. These results provide a mechanistic basis for FGFR4-R388 function in cancer invasion.

Independent regulation of short and long forms of latent TGF-beta binding protein (LTBP)-4 in cultured fibroblasts and human tissues.

Transforming growth factor (TGF)-beta is secreted and targeted into the extracellular matrix (ECM) in association with one of the latent TGF-beta binding proteins (LTBPs). Activation of these latent complexes is an important regulatory step in TGF-beta signaling. LTBPs target the growth factor into the ECM and expose it to activating mechanisms. Disruption of LTBP-4 gene causes severe developmental abnormalities in both humans and mice. Transcripts for two N-terminally distinct LTBP-4 variants, LTBP-4S (short) and -4L (long), have been identified. In the current work, we have characterized differences in the expression, processing, and ECM targeting of these LTBP-4 variants. Heart and skeletal muscle displayed expression of both variants, while liver expressed mainly LTBP-4L and lung as well as small intestine LTBP-4S. This tissue-specific expression pattern was found to originate from control of transcription by two independent promoters. Furthermore, LTBP-4S and -4L proteins were secreted and processed differently. During secretion, LTBP-4L was complexed with TGF-beta1, whereas the majority of LTBP-4S was secreted in a free form. In addition, LTBP-4S was incorporated into the ECM, while full-length LTBP-4L was not readily detectable in the ECM. These data suggest that LTBP-4 functions are modified by tissue-specific expression of the two N-terminally distinct variants, which in addition exhibit significant differences in cellular processing and targeting, that is, this provides a basis for understanding molecular diversity in LTBP-4 structure and function.

Inactivation of sestrin 2 induces TGF-beta signaling and partially rescues pulmonary emphysema in a mouse model of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-beta binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-beta and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.