thymosin-beta(4) and Melanoma

Thymosin β4 (Tβ4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tβ4 levels. Studies on stable clones with silenced

Topics: Actin Cytoskeleton; Biomarkers, Tumor; Biomechanical Phenomena; Carcinogenesis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Shape; Gene Silencing; Humans; Intermediate Filaments; Melanoma; Models, Biological; Neoplastic Stem Cells; Nestin; Thymosin; Vimentin

Melanoma is a malignancy characterized by high invasive/metastatic potential, with no efficient therapy after metastasis. Understanding the molecular mechanisms underlying the invasive/metastatic tendency is therefore important. Our genome-wide gene expression analyses revealed that human melanoma cell lines WM793 and especially WM239 (vertical growth phase and metastatic cells, respectively) overexpress the extracellular matrix (ECM) protein transforming growth factor β induced (TGFBI). In adhesion assays, recombinant TGFBI was strongly anti-adhesive for both melanoma cells and skin fibroblasts. TGFBI further impaired the adhesion of melanoma cells to the adhesive ECM proteins fibronectin, collagen-I, and laminin, known to interact with it. Unexpectedly, WM239 cells migrated/invaded more effectively in three-dimensional collagen-I and Matrigel cultures after knockdown of TGFBI by shRNA expression. However, in the physiological subcutaneous microenvironment in nude mice, after TGFBI knockdown, these cells showed markedly impaired tumor growth and invasive capability; the initially formed small tumors later underwent myxoid degeneration and completely regressed. By contrast, the expanding control tumors showed intense TGFBI staining at the tumor edges, co-localizing with the fibrillar fibronectin/tenascin-C/periostin structures that characteristically surround melanoma cells at invasion fronts. Furthermore, TGFBI was found in similar fibrillar structures in clinical human melanoma metastases as well, co-localizing with fibronectin. These data imply an important role for TGFBI in the ECM deposition and invasive growth of melanoma cells, rendering TGFBI a potential target for therapeutic interventions.

Topics: Actin Cytoskeleton; Animals; Cell Adhesion; Cell Movement; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genome-Wide Association Study; Humans; Integrin beta1; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Transplantation; Recombinant Proteins; Skin; Talin; Thymosin; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Thymosin beta4 (Tbeta4) is a major actin-sequestering protein that has been implicated in the growth, survival, motility, and metastasis of certain tumors and is considered an indicator for malignant progression. Therefore, identifying compounds that can downregulate Tbeta4 expression is very important for the development of anti-cancer chemotherapies. In this study, we investigated the effects of elevated cAMP on Tbeta4 expression and the metastatic potential of murine B16 melanoma cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tbeta4 suppression. We found that treatment with the cAMP-inducing compounds alpha-MSH (alpha-melanocyte stimulating hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tbeta4 expression and regulated EMT-associated genes through the suppression of NF-kappaB activation in B16F10 cells. Along with decreased Tbeta4 expression, the in vitro invasiveness and anchorage-independent growth in a semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of the alpha-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tbeta4 expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover, suppression of NF-kappaB activation by alpha-MSH or IBMX is critical for inhibiting Tbeta4 expression.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cyclic AMP; Gene Expression Regulation, Neoplastic; Matrix Metalloproteinase 9; Melanoma; Melanoma, Experimental; Mice; Neoplasm Metastasis; NF-kappa B; Thymosin

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the synthesis of polyamines essential for cell growth and proliferation. Its overexpression induces the transformation of murine fibroblasts in both sense and antisense orientations, yielding highly invasive tumors in nude mice. These cell lines hence provide a good model to study cell invasion. Here, the gene expression profiles of these cells were compared with their normal counterpart by microarray analyses (Incyte Genomics, Palo Alto, CA, and Affymetrix, Santa Clara, CA). Up-regulation of the actin sequestering molecule thymosin beta4 was the most prominent change in both cell lines. Tetracycline-inducible expression of thymosin beta4 antisense RNA caused a partial reversal of the transformed phenotype. Further, reversal of transformation by dominant-negative mutant of c-Jun (TAM67) caused reduction in thymosin beta4 mRNA. Interestingly, a sponge toxin, latrunculin A, which inhibits the binding of thymosin beta4 to actin, was found to profoundly affect the morphology and proliferation of the AdoMetDC transformants and to block their invasion in three-dimensional Matrigel. Thus, thymosin beta4 is a determinant of AdoMetDC-induced transformed phenotype and invasiveness. Up-regulation of thymosin beta4 was also found in ras-transformed fibroblasts and metastatic human melanoma cells. These data encourage testing latrunculin A-like and other agents interfering with thymosin beta4 for treatment of thymosin beta4-overexpressing tumors with high invasive and metastatic potential.

Topics: Adenosylmethionine Decarboxylase; Animals; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression Profiling; Humans; Melanoma; Mice; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Phenotype; Skin Neoplasms; Thymosin; Up-Regulation

Topics: Animals; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; ras Proteins; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; Thymosin; Tumor Cells, Cultured; Up-Regulation

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.

Topics: Animals; Fibronectins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Mice, Nude; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; ras Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein; Thymosin; Tumor Cells, Cultured