Establishment of Structure-Function Relationship of Tissue Inhibitor of Metalloproteinase-1 for Its Interaction with CD63: Implication for Cancer Therapy.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a pleiotropic protein, promoting both tumor-suppressive and tumor-promoting activities. While TIMP-1 is primarily known as an endogenous inhibitor of matrix metalloproteinases (MMPs) and thus associated with tumor cell invasion, clinical studies demonstrated increased expression of TIMP-1 and its association with poor prognosis in cancer. Non-MMP-inhibitory and oncogenic functions of TIMP-1 are mediated by induction of intracellular signaling via its cell surface receptor CD63, a tetraspanin. The present study investigates the structure-function relationship of TIMP-1 for its interaction with CD63, which may eventually help design a novel approach for targeting TIMP-1's pro-oncogenic activity without interfering its tumor suppressive MMP-inhibitory function. Importantly, our analysis includes TIMP-1/CD63 interactions at the cell surface of live cells. Here, we demonstrate that the 9 C-terminal amino acid residues of TIMP-1 and the large extracellular loop of CD63 are required for their interaction. Considering that the N-terminal half of TIMP-1 is sufficient for TIMP-1's MMP-inhibitory activity, we propose that those C-terminal amino acid residues are a potentially targetable motif of TIMP-1 oncogenic activity. As a proof of concept, we present the potential for the development of neutralizing antibodies against the C-terminal motif of TIMP-1 for disruption of TIMP-1 interaction with CD63 and the subsequent signal transduction.

An oncogenic activity of PDGF-C and its splice variant in human breast cancer.

Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer.

HPV-associated differential regulation of tumor metabolism in oropharyngeal head and neck cancer.

HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. The goal of the present study is to investigate the molecular mechanisms underlying the differential radiation sensitivity between HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC). Here, we show that HPV-negative HNSCC cells exhibit increased glucose metabolism as evidenced by increased production of lactate, while HPV-positive HNSCC cells effectively utilize mitochondrial respiration as evidenced by increased oxygen consumption. HPV-negative cells express HIF1α and its downstream mediators of glucose metabolism such as hexokinase II (HKII) and carbonic anhydrase IX (CAIX) at higher levels, while the expression level of cytochrome c oxidase (COX) was noticeably higher in HPV-positive HNSCC. In addition, the expression levels of pyruvate dehydrogenase kinases (PDKs), which inhibit pyruvate dehydrogenase activity, thereby preventing entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle, were much higher in HPV-negative HNSCC compared to those in HPV-positive cells. Importantly, a PDK inhibitor, dichloroacetate, effectively sensitized HPV-negative cells to irradiation. Lastly, we found positive interactions between tonsil location and HPV positivity for COX intensity and COX/HKII index ratio as determined by immunohistochemical analysis. Overall survival of patients with HNSCC at the tonsil was significantly improved with an increased COX expression. Taken together, the present study provides molecular insights into the mechanistic basis for the differential responses to radiotherapy between HPV-driven vs. spontaneous or chemically induced oropharyngeal cancer.

Dynamic regulation of platelet-derived growth factor D (PDGF-D) activity and extracellular spatial distribution by matriptase-mediated proteolysis.

The oncogenic roles of PDGF-D and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. Latent full-length PDGF-D (FL-D) consists of a CUB domain, a growth factor domain (GFD), and the hinge region in between. Matriptase processes the FL-D dimer into a GFD dimer (GFD-D) in a stepwise manner, involving generation of a hemidimer (HD), an intermediate product containing one FL-D subunit and one GFD subunit. Although the HD is a pro-growth factor that can be processed into the GFD-D by matriptase, the HD can also act as a dominant-negative ligand that prevents PDGF-B-mediated ß-PDGF receptor activation in fibroblasts. The active GFD-D can be further cleaved into a smaller and yet inactive form if matriptase-mediated proteolysis persists. Through mutagenesis and functional analyses, we found that the R(340)R(341)GR(343)A (P4-P1/P1') motif within the GFD is the matriptase cleavage site through which matriptase can deactivate PDGF-D. Comparative sequence analysis based on the published crystal structure of PDGF-B predicted that the matriptase cleavage site R(340)R(341)GR(343)A is within loop III of the GFD, a critical structural element for its binding with the ß-PDGF receptor. Interestingly, we also found that matriptase processing regulates the deposition of PDGF-D dimer species into the extracellular matrix (ECM) with increased binding from the FL-D dimer, to the HD, and to the GFD-D. Furthermore, we provide evidence that R(340)R(341)GR(343)A within the GFD is critical for PDGF-D deposition and binding to the ECM. In this study, we report a structural element crucial for the biological function and ECM deposition of PDGF-D and provide molecular insight into the dynamic functional interplay between the serine protease matriptase and PDGF-D.

Discoidin domain receptor 1 is a novel transcriptional target of ZEB1 in breast epithelial cells undergoing H-Ras-induced epithelial to mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) process allows carcinoma cells to dissociate from the primary tumor thereby facilitating tumor cell invasion and metastasis. Ras-dependent hyperactive signaling is commonly associated with tumorigenesis, invasion, EMT, and metastasis. However, the downstream effectors by which Ras regulates EMT remain ill defined. In this study, we show that the H-Ras pathway leads to mesenchymal-like phenotypic changes in human breast epithelial cells by controlling the ZEB1/microRNA-200c axis. Moreover, H-Ras suppresses the expression of the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, via ZEB1, thus identifying ZEB1 as a novel transcriptional repressor of DDR1. Mutation studies on the putative promoter of the DDR1 gene revealed that bipartite Z- and E-box elements play a key role in transcriptional repression of DDR1 in Hs578T and MDA-MB-231 breast carcinoma cell lines by ZEB1. Furthermore, we found an inverse correlation between ZEB1 and DDR1 expression in various cancer cell lines and in human breast carcinoma tissues. Consistently, overexpression of DDR1 reduced the invasive phenotype of mesenchymal-like triple-negative breast cancer cells in 3D cultures and in vivo. Thus, ZEB1's role in maintenance of EMT in breast carcinoma cells is mediated in part by its ability to suppress DDR1 expression and consequently contribute to the activation of the invasive phenotype. Taken together, our results unveil a novel H-Ras/ZEB1/DDR1 network that contributes to breast cancer progression in triple-negative breast cancers.

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth.

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis.

TIMP-1 via TWIST1 induces EMT phenotypes in human breast epithelial cells.

UNLABELLED: Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates intracellular signaling networks for inhibition of apoptosis. Tetraspanin (CD63), a cell surface binding partner for TIMP-1, was previously shown to regulate integrin-mediated survival pathways in the human breast epithelial cell line MCF10A. In the current study, we show that TIMP-1 expression induces phenotypic changes in cell morphology, cell adhesion, cytoskeletal remodeling, and motility, indicative of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecule E-cadherin with an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Signaling through TIMP-1, but not TIMP-2, induces the expression of TWIST1, an important EMT transcription factor known to suppress E-cadherin transcription, in a CD63-dependent manner. RNAi-mediated knockdown of TWIST1 rescued E-cadherin expression in TIMP-1-overexpressing cells, demonstrating a functional significance of TWIST1 in TIMP-1-mediated EMT. Furthermore, analysis of TIMP-1 structural mutants reveals that TIMP-1 interactions with CD63 that activate cell survival signaling and EMT do not require the matrix metalloproteinase (MMP)-inhibitory domain of TIMP-1. Taken together, these data demonstrate that TIMP-1 binding to CD63 activates intracellular signal transduction pathways, resulting in EMT-like changes in breast epithelial cells, independent of its MMP-inhibitory function. IMPLICATIONS: TIMP-1's function as an endogenous inhibitor of MMP or as a "cytokine-like" signaling molecule may be a critical determinant for tumor cell behavior.

A critical role of the PTEN/PDGF signaling network for the regulation of radiosensitivity in adenocarcinoma of the prostate.

PURPOSE: Loss or mutation of the phosphate and tensin homologue (PTEN) is a common genetic abnormality in prostate cancer (PCa) and induces platelet-derived growth factor D (PDGF D) signaling. We examined the role of the PTEN/PDGF axis on radioresponse using a murine PTEN null prostate epithelial cell model. METHODS AND MATERIALS: PTEN wild-type (PTEN+/+) and PTEN knockout (PTEN-/-) murine prostate epithelial cell lines were used to examine the relationship between the PTEN status and radiosensitivity and also to modulate the PDGF D expression levels. PTEN-/- cells were transduced with a small hairpin RNA (shRNA) lentiviral vector containing either scrambled nucleotides (SCRM) or sequences targeted to PDGF D (shPDGF D). Tumorigenesis and morphogenesis of these cell lines were evaluated in vivo via subcutaneous injection of male nude mice and in vitro using Matrigel 3-dimensional (3D) culture. Effects of irradiation on clonogenic survival, cell migration, and invasion were measured with respect to the PTEN status and the PDGF D expression level. In addition, apoptosis and cell cycle redistribution were examined as potential mechanisms for differences seen. RESULTS: PTEN-/- cells were highly tumorigenic in animals and effectively formed foci in 3D culture. Importantly, loss of PDGF D in these cell lines drastically diminished these phenotypes. Furthermore, PTEN-/- cells demonstrated increased clonogenic survival in vitro compared to PTEN+/+, and attenuation of PDGF D significantly reversed this radioresistant phenotype. PTEN-/- cells displayed greater migratory and invasive potential at baseline as well as after irradiation. Both the basal and radiation-induced migratory and invasive phenotypes in PTEN-/- cells required PDGF D expression. Interestingly, these differences were independent of apoptosis and cell cycle redistribution, as they showed no significant difference. CONCLUSIONS: We propose that PDGF D represents a potentially promising target for PCa treatment resistance in the absence of PTEN function, and warrants further laboratory evaluation and clinical study.

A novel function of HPV16-E6/E7 in epithelial-mesenchymal transition.

Human papillomavirus (HPV) 16 is among the most important etiological factors in many human cancers, including head and neck squamous cell carcinomas (HNSCCs) not associated with alcohol or tobacco use. HPV16-E6 and E7 oncoproteins target intracellular signaling networks, altering key molecular and cellular events during tumor progression. The present study investigates the role of HPV16-E6 and E7 oncogenes on the epithelial-mesenchymal transition (EMT), a cellular process thought to be critical for tumor cell invasion and metastasis. Using the epithelial MDCK cell line as an in vitro model, we show that the stable expression of HPV16-E6 or E7 induces morphological conversion from cobblestone-shaped epithelium to spindle-shaped mesenchyme-like phenotype. Consistent with these morphological changes, both E6 and E7 induce expression of the EMT-activating transcriptional factors Slug, Twist, ZEB1 and ZEB2, especially ZEBs, accompanied with switch from epithelial to mesenchymal markers. Importantly, E6 and E7 expression results in induction of the migratory and invasive potential, a functional hallmark of EMT. When we examined the association between HPV16 and the EMT signature in HNSCC cell lines derived from head and neck cancer patients, we found a correlation between HPV16 positivity and the expression of EMT transcription factor ZEB1. Taken together, our findings suggest HPV16 induces EMT-like processes via induction of the EMT transcription factors which may contribute to tumor progression and metastasis.

TIMP-1 induces an EMT-like phenotypic conversion in MDCK cells independent of its MMP-inhibitory domain.

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1's ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression.

PDGF upregulates Mcl-1 through activation of ß-catenin and HIF-1α-dependent signaling in human prostate cancer cells.

BACKGROUND: Aberrant platelet derived growth factor (PDGF) signaling has been associated with prostate cancer (PCa) progression. However, its role in the regulation of PCa cell growth and survival has not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: Using experimental models that closely mimic clinical pathophysiology of PCa progression, we demonstrated that PDGF is a survival factor in PCa cells through upregulation of myeloid cell leukemia-1 (Mcl-1). PDGF treatment induced rapid nuclear translocation of ß-catenin, presumably mediated by c-Abl and p68 signaling. Intriguingly, PDGF promoted formation of a nuclear transcriptional complex consisting of ß-catenin and hypoxia-inducible factor (HIF)-1α, and its binding to Mcl-1 promoter. Deletion of a putative hypoxia response element (HRE) within the Mcl-1 promoter attenuated PDGF effects on Mcl-1 expression. Blockade of PDGF receptor (PDGFR) signaling with a pharmacological inhibitor AG-17 abrogated PDGF induction of Mcl-1, and induced apoptosis in metastatic PCa cells. CONCLUSIONS/SIGNIFICANCE: Our study elucidated a crucial survival mechanism in PCa cells, indicating that interruption of the PDGF-Mcl-1 survival signal may provide a novel strategy for treating PCa metastasis.

Cediranib inhibits both the intraosseous growth of PDGF D-positive prostate cancer cells and the associated bone reaction.

BACKGROUND: The major cause of death in prostate cancer (PCa) cases is due to distant metastatic lesions, with the bone being the most prevalent site for secondary colonization. Utilization of small molecule inhibitors to treat bone metastatic PCa have had limited success either as monotherapies or in combination with other chemotherapeutics due to intolerable toxicities. In the current study, we developed a clinically relevant in vivo intraosseous tumor model overexpressing the platelet-derived growth factor D (PDGF D) to test the efficacy of a newly characterized vascular endothelial growth factor receptor (VEGFR)/PDGFR inhibitor, cediranib (also called AZD2171). METHODS: An intratibial-injection model was established utilizing DU145 cells with or without increased PDGF D expression. Tumor-bearing mice were treated by daily gavage administration of cediranib and/or weekly i.p. injection of docetaxel for 7 weeks. Tibiae were monitored by in vivo/ex vivo X-rays and histomorphometry analysis was performed to estimate tumor volume and tumor-associated trabecular bone growth. RESULTS: Cediranib reduced intraosseous growth of prostate tumors as well as tumor-associated bone responses. When compared to the standard chemotherapeutic agent docetaxel, cediranib exhibited a stronger inhibition of tumor-associated bone response. The efficacy of cediranib was further enhanced when the drug was co-administered with docetaxel. Importantly, the therapeutic benefits of cediranib and docetaxel are more prominent in intraosseous prostate tumors overexpressing PDGF D. CONCLUSION: These novel findings support the utilization of cediranib, either alone or in combination with docetaxel, to treat bone metastatic PCa exhibiting PDGF D expression.

PTEN regulates PDGF ligand switch for ß-PDGFR signaling in prostate cancer.

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor ß (ß-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for ß-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and ß-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and ß-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/ß-PDGFR signal transduction.

Platelet-derived growth factor-C (PDGF-C) activation by serine proteases: implications for breast cancer progression.

The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.

Identification of H-Ras-specific motif for the activation of invasive signaling program in human breast epithelial cells.

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

A novel signaling axis of matriptase/PDGF-D/ß-PDGFR in human prostate cancer.

Increasing evidence indicates the significance of platelet-derived growth factor receptor-ß (ß-PDGFR) signaling in prostate cancer (PCa). Accordingly, preclinical studies suggest the potential of ß-PDGFR as a therapeutic target in metastatic PCa. However, a ligand responsible for ß-PDGFR activation in PCa was unknown, and recent clinical trials with imatinib mesylate showed limited success due to normal tissue toxicity. Similarly, in spite of mounting evidence indicating the significance of matriptase in PCa, little is known about its substrates or molecular actions during PCa progression. Here, we identified PDGF-D as a ligand for ß-PDGFR in PCa and discovered matriptase as its regulator. Matriptase activates PDGF-D by proteolytic removal of the CUB domain in a 2-step process, creating a hemidimer, followed by growth factor domain dimer (GFD-D) generation. Matriptase can deactivate PDGF-D by further proteolytic cleavage within the GFD, revealing its biphasic regulation. Importantly, PDGF-D/matriptase colocalization is accompanied with ß-PDGFR phosphorylation in human PCa tissues. This study unveiled a novel signaling axis of matriptase/PDGF-D/ß-PDGFR in PCa, providing new insights into functional interplay between serine protease and growth factor signaling networks.

Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP.

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN(+/-)) or homozygous (PTEN(-/-)) for PTEN deletion or harboring a wild-type PTEN (PTEN(+/+)) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN(-/-) PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN(+/+) and PTEN(+/-) cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN(-/-) cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN(-/-) cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN(+/+) cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.

Shedding of RANKL by tumor-associated MT1-MMP activates Src-dependent prostate cancer cell migration.

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an essential role in protease-mediated extracellular matrix (ECM) degradation, but it also functions as a sheddase releasing non-ECM substrates such as receptor activator of NF-kappaB ligand (RANKL), an osteoclastogenic factor typically confined to the surface of osteoblasts. We previously found high expression of MT1-MMP in skeletal metastasis of prostate cancer patients, in a pattern similar to RANKL expression. We also showed that overexpression of MT1-MMP in prostate cancer cells increases tumor growth and osteolysis in an intratibial mouse model of bone metastasis, and that soluble factor(s) shed by tumor-derived MT1-MMP enhance osteoclast differentiation in a RANKL-dependent manner. Recent evidence indicates that the cognate receptor for RANKL, RANK, is expressed in prostate cancer cells, suggesting the presence of an autocrine pathway. In this study, we show that MT1-MMP-expressing LNCaP prostate cancer cells display enhanced migration. Moreover, conditioned medium from LNCaP cells expressing both RANKL and MT1-MMP stimulates the migration of MT1-MMP-deficient C42b prostate cancer cells. This enhanced chemotaxis can be abrogated by osteoprotegerin (soluble decoy receptor of RANKL), MIK-G2 (a selective inhibitor for MT1-MMP), and PP2 (a Src inhibitor). These findings indicate that tumor-derived MT1-MMP enhances tumor cell migration through initiation of an autocrine loop requiring ectodomain shedding of membrane-bound RANKL in prostate cancer cells, and that Src is a key downstream mediator of RANKL-induced migration of prostate cancer cells.

miR-200 regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells.

MicroRNAs have been implicated in tumor progression. Recent studies have shown that the 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 suggests that the processes of EMT can be triggered by various growth factors, such as transforming growth factor beta and platelet-derived growth factor-D (PDGF-D). Moreover, we recently reported that overexpression of PDGF-D in prostate cancer cells (PC3 PDGF-D cells) leads to the acquisition of the 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 downregulation of the miR-200 family in PC3 PDGF-D cells as well as in PC3 cells exposed to purified active PDGF-D protein, resulting in the upregulation of ZEB1, ZEB2, and Snail2 expression. Interestingly, re-expression of miR-200b in PC3 PDGF-D cells led to reversal of the EMT phenotype, which was associated with the downregulation of ZEB1, ZEB2, and Snail2 expression, and these results were consistent with greater expression levels 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 the culture surface and cell detachment. From these results, we conclude that PDGF-D-induced acquisition of the EMT phenotype in PC3 cells is, in part, a result of repression of miR-200 and that any novel strategy by which miR-200 could be upregulated would become a promising approach for the treatment of invasive prostate cancer.

Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion.

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.