RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.     

S100A8/A9 activate key genes and pathways in colon tumor progression

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.     

Interaction of extracellular S100A4 with RAGE prompts prometastatic activation of A375 melanoma cells

S100A4, a member of the S100 protein family of EF‐hand calcium‐binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild‐type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375‐hS100A4) or human receptor for advanced glycation endproducts (A375‐hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum‐Golgi‐dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4‐RAGE interaction and its blockade on A375, A375‐hS100A4, A375‐hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.     

In vivo overexpression of tumstatin domains by tumor cells inhibits their invasive properties in a mouse melanoma model

Our previous studies demonstrated that a synthetic peptide encompassing residues 185-203 of the noncollagenous (NC1) domain of the alpha3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1-232) or its C-terminal part, encompassing residues 185-203 (Tum 183-232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by -60% and -56%, respectively, with B16F1 cells overexpressing Tum 1-232 or Tum 183-232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1-232 or Tum 183-232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183-232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism.     

The amphoterin (HMGB1)/receptor for advanced glycation end products (RAGE) pair modulates myoblast proliferation, apoptosis, adhesiveness, migration, and invasiveness. Functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGE delta cyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mock-transfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21(Waf1), and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGE delta cyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGE delta cyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation.     

S100A9 interaction with TLR4 promotes tumor growth

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.     

S100P stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE)

S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-kappaB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival.     

Bcl-2 overexpression in melanoma cells increases tumor progression-associated properties and in vivo tumor growth

In this study, we demonstrated that bcl-2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis-related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl-2 overexpressing derivatives, we found that bcl-2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP-2, MMP-7, MT1-MMP, and tissue inhibitors of metalloproteases-1 and -2. Moreover, bcl-2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl-2 transfectants tumors is significantly associated to an increase in MMP-2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl-2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression.     

Arginase-II promotes melanoma migration and adhesion through enhancing hydrogen peroxide production and STAT3 signaling

Elevated arginase type II (Arg-II) associates with higher grade tumors. Its function and underlying molecular mechanisms in melanoma remain elusive. In the present study, we observed a significantly higher frequency of Arg-II expression in melanoma of patients with metastasis than those without metastasis. Silencing Arg-II in two human melanoma cell lines slowed down the cell growth, while overexpression of native but not a catalytically inactive Arg-II promoted cell proliferation without affecting cell death. Treatment of cells with arginase inhibitor also reduced melanoma cell number, demonstrating that Arg-II promotes melanoma cell proliferation dependently of its enzymatic activity. However, results from silencing Arg-II or overexpressing native or the inactive Arg-II as well as treatment with arginase inhibitor showed that Arg-II promotes melanoma metastasis-related processes, such as melanoma cell migration and adhesion on endothelial cells, independently of its enzymatic activity. Moreover, the treatment of the cells with STAT3 inhibitor suppressed Arg-II-promoted melanoma cell migration and adhesion. Furthermore, catalase, but not superoxide dismutase, prevented STAT3 activation as well as increased melanoma cell migration and adhesion induced by overexpressing native or the inactive Arg-II. Taken together, our study uncovers both activity-dependent and independent mechanisms of Arg-II in promoting melanoma progression. While Arg-II enhances melanoma cell proliferation through polyamine dependently of its enzymatic activity, it promotes metastasis-related processes, that is, migration and adhesion onto endothelial cell, through mitochondrial H₂O₂-STAT3 pathway independently of the enzymatic activity. Suppressing Arg-II expression rather than inhibiting its enzymatic activity may, therefore, represent a novel strategy for the treatment of melanoma.     

Cell migration or cytokinesis and proliferation? – Revisiting the “go or grow” hypothesis in cancer cells in vitro

The mortality of patients with solid tumors is mostly due to metastasis that relies on the interplay between migration and proliferation. The “go or grow” hypothesis postulates that migration and proliferation spatiotemporally excludes each other.We evaluated this hypothesis on 35 cell lines (12 mesothelioma, 13 melanoma and 10 lung cancer) on both the individual cell and population levels. Following three-day-long videomicroscopy, migration, proliferation and cytokinesis-length were quantified. We found a significantly higher migration in mesothelioma cells compared to melanoma and lung cancer while tumor types did not differ in mean proliferation or duration of cytokinesis. Strikingly, we found in melanoma and lung cancer a significant positive correlation between mean proliferation and migration. Furthermore, non-dividing melanoma and lung cancer cells displayed slower migration. In contrast, in mesothelioma there were no such correlations. Interestingly, negative correlation was found between cytokinesis-length and migration in melanoma. FAK activation was higher in melanoma cells with high motility.We demonstrate that the cancer cells studied do not defer proliferation for migration. Of note, tumor cells from various organ systems may differently regulate migration and proliferation. Furthermore, our data is in line with the observation of pathologists that highly proliferative tumors are often highly invasive.     

T-cadherin suppresses the cell proliferation of mouse melanoma B16F10 and tumor angiogenesis in the model of the chorioallantoic membrane

The influence of T-cadherin on the pigmentation and proliferation of mouse melanoma B16F10 cells in vitro and on the growth and neovascularization of tumor cell masses formed by the B16F10 cells in a model of the chorioallantoic membrane of a chicken embryo is studied. It is found that the proliferative activity of the cells decreases in the cell culture of mouse melanoma upon the overexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the overexpression of T-cadherin are rarely settling are to the chorioallantoic membrane than the control cells. In addition, it is found that the control cells of mouse melanoma form tumors with area more 0.1 mm² more often than the cells with the overexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the overexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the overexpression of T-cadherin in the B16F10 cells suppresses the proliferation of these cells in vitro and the growth of the tumor masses formed by melanoma cells on the chorioallantoic membrane and their neovascularization in vivo.     

Immunohistochemical Expression of RAGE and Its Ligand (S100A9) in Cervical Lesions

Altered expressions of receptor for advanced glycation end-products (RAGE) and its ligand (S100A9) are observed in many cancers and play a key role in inflammation-associated cancer. In our previous study, by two-dimensional gel electrophoresis followed by mass spectrometry, the expression of S100A9 protein was found to increase in squamous cervical cancer compared with adjacent normal cervical tissues. Therefore, in the present study we observed the expressions of S100A9 and RAGE in 30 chronic cervicitis, 50 cervical intraepithelial neoplasia (CIN), and 40 squamous cervical cancer (SCC) using immunohistochemical analysis and analyzed the differential expression and possible role of S100A9 and RAGE in cancer development. Immunohistochemical findings were as follows: the expressions of S100A9 and RAGE were demonstrated in chronic cervicitis, CIN, and SCC. Moreover, their expressions were gradually increasing as the tumor progressed. In SCC, the staining scores of S100A9 and RAGE were significantly higher in well-differentiated tumors compared to moderately and poorly differentiated tumors. The expression of S100A9 in epithelial cells exhibited a positive correlation to RAGE expression in chronic cervicitis, CIN, and SCC. There were no significant difference of S100A9 immunoreactivity in stromal cells among chronic cervicitis, CIN, and SCC. Moreover, there was no correlation between S100A9 immunoreactivity in stromal cells of SCC and clinicopathological parameters. Finally, double immunohistochemistry illustrated that RAGE and S100A9 co-express in SCC. In conclusion, RAGE binds its ligand (S100A9), which plays an important role in the development of SCC. In addition, the expressions of S100A9 and RAGE in SCC tumor cells were closely associated with histological differentiation.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

S100A14 stimulates cell proliferation and induces cell apoptosis at different concentrations via receptor for advanced glycation end products (RAGE)

S100A14 is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effects on different types of cells. However, exact extracellular roles of S100A14 have not been clarified yet. Here we investigated the effects of S100A14 on esophageal squamous cell carcinoma (ESCC) cell lines. Results demonstrated that low doses of extracellular S100A14 stimulate cell proliferation and promote survival in KYSE180 cells through activating ERK1/2 MAPK and NF-κB signaling pathways. Immunoprecipitation assay showed that S100A14 binds to receptor for advanced glycation end products (RAGE) in KYSE180 cells. Inhibition of RAGE signaling by different approaches including siRNA for RAGE, overexpression of a dominant-negative RAGE construct or a RAGE antagonist peptide (AmphP) significantly blocked S100A14-induced effects, suggesting that S100A14 acts via RAGE ligation. Furthermore, mutation of the N-EF hand of S100A14 (E39A, E45A) virtually reduced 10 μg/ml S100A14-induced cell proliferation and ERK1/2 activation. However, high dose (80 μg/ml) of S100A14 causes apoptosis via the mitochondrial pathway with activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase. High dose S100A14 induces cell apoptosis is partially in a RAGE-dependent manner. This is the first study to demonstrate that S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades, promoting cell proliferation or triggering cell apoptosis at different doses.     

Propofol suppresses proliferation,invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.     

CSMD1 exhibits antitumor activity in A375 melanoma cells through activation of the Smad pathway

In this work, we studied the effects of CUB and Sushi multiple domains 1 gene (CSMD1) expression in A375 melanoma cells in vivo and in vitro. CSDM1 expression decreased proliferation and migration, and increased apoptosis and G(1) arrest in A375 cells in vitro. Expression of CSDM1 in established xenografted tumors decreased tumor size and weight, and decreased the density of intratumor microvessels. The survival rate of mice with tumors expressing CSMD1 was significantly higher than mice with tumors that did not express CSDM1. These results confirm the role of CSDM1 as a tumor suppressor gene in melanoma cells. Furthermore, we found that CSMD1 can interact with Smad3, activate Smad1, Smad2, and Smad3, and increase the expression of Smad4. These results might prove helpful for the development of novel therapies for melanoma treatment.