Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases (MMPs) and the balance between MMPs/TIMPs regulates the extracellular matrix (ECM) turnover and remodeling during normal development and pathogenesis. Increasing evidence indicates a much more complex role for TIMPs during tumor progression and angiogenesis, in addition to their regulation of MMP-mediated ECM degradation. In this article, we review both the MMP-dependent and -independent actions of TIMPs for the regulation of cell death, cell proliferation, and angiogenesis, with a particular emphasis on TIMP-1 in the regulation of tetraspanin/integrin-mediated cell survival signal transduction pathways.
This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast twohybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin b1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin b1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 colocalization with integrin b1, and consequently reversed TIMP-1-mediated integrin b1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.
MicroRNAs have been implicated in tumor progression. Recent studies have shown that miR-200 family regulates Epithelial-Mesenchymal Transition (EMT) by targeting zinc-finger E-box binding homeobox 1 (ZEB1) and ZEB2. Emerging evidence from our laboratory and others suggest that the processes of EMT can be triggered by various growth factors such as Transforming Growth Factor-beta (TGF-β) and Platelet-Derived Growth Factor-D (PDGF-D). Moreover, we have recently reported that over-expression of PDGF-D in prostate cancer cells (PC3 PDGF-D cells) leads to the acquisition of EMT phenotype, and this model offers an opportunity for investigating the molecular interplay between PDGF-D signaling and EMT. Here we report, for the first time, significant down-regulation of miR-200 family in PC3 PDGF-D cells as well as in PC3 cells exposed to purified active PDGF-D protein, resulting in the up-regulation of ZEB1, ZEB2 and snail2 expression. Interestingly, re-expression of miR-200b in PC3 PDGF-D cells led to the reversal of EMT phenotype, which was associated with the down-regulation of ZEB1, ZEB2 and snail2 expression and these results were consistent with increased gene expressions of epithelial markers. Moreover, transfection of PC3 PDGF-D cells with miR-200b inhibited cell migration and invasion with concomitant repression of cell adhesion to culture surface and cell detachment. From these results, we conclude that PDGF-D induced acquisition of EMT phenotype of PC3 cells is in part due to repression of miR-200 and that any novel strategies by which miR-200 could be up-regulated would become a promising approach for the treatment of invasive prostate cancer.
Galectin-3 is a multifunctional oncogenic protein found in the nucleus and cytoplasm and also the extracellular milieu. Although recent studies demonstrated an anti-apoptotic activity of galectin-3, neither the functional site nor the mechanism of how galectin-3 regulates apoptosis is known. In this study, we examined the subcellular localization of galectin-3 during apoptosis and investigated its anti-apoptotic actions. We report that galectin-3 translocates to the perinuclear membrane following a variety of apoptotic stimuli. Confocal microscopy and biochemical analysis revealed that galectin-3 is enriched in the mitochondria and prevents mitochondrial damage and cytochrome c release. Using a yeast two-hybrid system, we screened for galectin-3-interacting proteins that regulate galectin-3 localization and anti-apoptotic activity. Synexin, a Ca 2؉ -and phospholipid-binding protein, was one of the proteins identified. We confirmed direct interaction between galectin-3 and synexin by glutathione S-transferase pulldown assay in vitro. We showed that galectin-3 failed to translocate to the perinuclear membranes when expression of synexin was down-regulated using an oligodeoxyribonucleotide complementary to the synexin mRNA, suggesting a role for synexin in galectin-3 trafficking. Furthermore, synexin down-regulation abolished anti-apoptotic activity of galectin-3. Taken together, these results suggest that synexin mediates galectin-3 translocation to the perinuclear mitochondrial membranes, where it regulates mitochondrial integrity critical for apoptosis regulation.Galectin-3 is a 31-kDa member of the -galactoside-binding family of proteins found widely in epithelial and immune cells. Expression of galectin-3 is associated with neoplastic progression and metastatic potential (1-5) in head and neck (6), thyroid (7), gastric (3), and colon (8) cancers, suggesting a role in oncogenesis. Galectin-3 modulates a variety of cellular processes. Extracellular galectin-3 mediates cell migration, cell adhesion, and cell/cell interactions, whereas nuclear galectin-3 is involved in pre-mRNA splicing (9 -11). Interestingly, recent studies showed that cytoplasmic, but not nuclear, galectin-3 is associated with tumor progression (12, 13). Yet, the role of cytoplasmic galectin-3 is unknown.We (15-17) and others (14,18,19) have previously shown that galectin-3 inhibits T-cell apoptosis induced by anti-Fas antibody and epithelial cell apoptosis induced by staurosporine, cisplatin, genistein, and anoikis. The anti-apoptotic activity of galectin-3 was also demonstrated in galectin-3-deficient mice. Peritoneal macrophages from galectin-3-deficient mice were more sensitive to apoptotic stimuli than those from control mice (20). The ability of galectin-3 to protect cells against apoptosis induced by agents working through different mechanisms suggests that galectin-3 regulates the common apoptosis commitment step.During the past decade, explosive progress has been made toward understanding the molecular basis for the regulation of the a...
Platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. The vital functions of PDGFs for angiogenesis, as well as development of kidney, brain, cardiovascular system and pulmonary alveoli during embryogenesis, have been well demonstrated by gene knock-out approaches. Clinical studies reveal that aberrant expression of PDGF and its receptor is often associated with a variety of disorders including atherosclerosis, fibroproliferative diseases of lungs, kidneys and joints, and neoplasia. PDGF contributes to cancer development and progression by both autocrine and paracrine signaling mechanisms. In this review article, important features of the PDGF isoforms and their cell surface receptor subunits are discussed, with regards to signal transduction, PDGF-isoform specific cellular responses, and involvement in angiogensis, and tumorstromal interactions.
Galectin-3, a -galactoside-binding protein, is implicated in cell growth, adhesion, differentiation, and tumor progression by interactions with its ligands. Recent studies have revealed that galectin-3 suppresses apoptosis and anoikis that contribute to cell survival during metastatic cascades. Previously, it has been shown that human galectin-3 undergoes post-translational signaling modification of Ser 6 phosphorylation that acts as an "on/off" switch for its sugar-binding capability. We questioned whether galectin-3 phosphorylation is required for its anti-apoptotic function. Serine to alanine (S6A) and serine to glutamic acid (S6E) mutations were produced at the casein kinase I phosphorylation site in galectin-3. The cDNAs were transfected into a breast carcinoma cell line BT-549 that innately expresses no galectin-3. Metabolic labeling revealed that only wild type galectin-3 undergoes phosphorylation in vivo. Expression of Ser 6 mutants of galectin-3 failed to protect cells from cisplatin-induced cell death and poly(ADPribose) polymerase from degradation when compared with wild type galectin-3. The non-phosphorylated galectin-3 mutants failed to protect cells from anoikis with G 1 arrest when cells were cultured in suspension. In response to a loss of cell-substrate interactions, only cells expressing wild type galectin-3 down-regulated cyclin A expression and up-regulated cyclin D 1 and cyclindependent kinase inhibitors, i.e. p21 WAF1/CIP1 and p27 KIP1 expression levels. These results demonstrate that galectin-3 phosphorylation regulates its anti-apoptotic signaling activity.
Tissue inhibitor of metalloproteinase (TIMP-1) is a natural protease inhibitor of matrix metalloproteinases (MMPs). Recent studies revealed a novel function of TIMP-1 as a potent inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not understood. Here we show that TIMP-1 activates cell survival signaling pathways involving focal adhesion kinase, phosphatidylinositol 3-kinase, and ERKs in human breast epithelial cells to TIMP-1. TIMP-1-activated cell survival signaling down-regulates caspase-mediated classical apoptotic pathways induced by a variety of stimuli including anoikis, staurosporine exposure, and growth factor withdrawal. Consistently, down-regulation of TIMP-1 expression greatly enhances apoptotic cell death. In a previous study, substitution of the second amino acid residue threonine for glycine in TIMP-1, which confers selective MMP inhibition, was shown to obliterate its anti-apoptotic activity in activated hepatic stellate cells suggesting that the anti-apoptotic activity of TIMP-1 is dependent on MMP inhibition. Here we show that the same mutant inhibits apoptosis of human breast epithelial cells, suggesting different mechanisms of TIMP-1 regulation of apoptosis depending on cell types. Neither TIMP-2 nor a synthetic MMP inhibitor protects breast epithelial cells from intrinsic apoptotic cell death. Furthermore, TIMP-1 enhances cell survival in the presence of the synthetic MMP inhibitor. Taken together, the present study unveils some of the mechanisms mediating the anti-apoptotic effects of TIMP-1 in human breast epithelial cells through TIMP-1-specific signal transduction pathways. Cell interactions with extracellular matrix (ECM)1 greatly influence cell survival, and removal of anchorage-dependent cells from their association with the ECM results in apoptotic cell death, known as anoikis (1, 2). Cell-ECM interaction-mediated signal transduction is regulated in part by the composition and integrity of the ECM and actions of its components on specific cell adhesion receptors (3-6). Integrity and turnover of the ECM are in part regulated by matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases (7-9) known to accomplish the degradation of ECM components. Four members of the tissue inhibitor of metalloproteinase family (TIMP-1 to -4) have been identified as natural inhibitors of MMPs.Previous studies in our laboratory (10) showed that bcl-2 overexpression is associated with enhanced levels of TIMP-1 expression in human breast epithelial cells, suggesting a role for TIMP-1 in apoptosis. Indeed, apoptosis studies showed that TIMP-1 protects against a variety of apoptotic stimuli including anoikis, hydrogen peroxide, x-ray irradiation, and adriamycin treatment (10). Furthermore, TIMP-1 inhibition of apoptosis in human breast epithelial cells involves focal adhesion kinase (FAK)-mediated cell survival signaling rather than regulation of cell-ECM interactions via MMP activity.During the past several years, investiga...
The majority of human malignancies are believed to have epithelial origin, and the progression of cancer is often associated with a transient process named epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of epithelial markers and the gain of mesenchymal markers that are typical of "cancer stem-like cells, " which results in increased cell invasion and metastasis in vivo. Therefore, it is important to uncover the mechanistic role of factors that may induce EMT in cancer progression. Studies have shown that platelet-derived growth factor (PDGF) signaling contributes to EMT, and more recently, PDGF-D has been shown to regulate cancer cell invasion and angiogenesis. However, the mechanism by which PDGF-D promotes invasion and metastases and whether it is due to the acquisition of EMT phenotype remain elusive. For this study, we established stably transfected PC3 cells expressing high levels of PDGF-D, which resulted in the significant induction of EMT as shown by changes in cellular morphology concomitant with the loss of E-cadherin and zonula occludens-1 and gain of vimentin. We also found activation of mammalian target of rapamycin and nuclear factor-B, as well as Bcl-2 overexpression, in PDGF-D PC3 cells, which was associated with enhanced adhesive and invasive behaviors. More importantly, PDGF-D-overexpressing PC3 cells showed tumor growth in SCID mice much more rapidly than PC3 cells. These results provided a novel mechanism by which PDGF-D promotes EMT, which in turn increases tumor growth, and these results further suggest that PDGF-D could be a novel therapeutic target for the prevention and/or treatment of prostate cancer.
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