Divergent roles of lysyl oxidase family members in ornithine decarboxylase- and RAS-transformed mouse fibroblasts and human melanoma cells.

We have previously shown that proto-oncoprotein c-Jun is activated in ornithine decarboxylase (ODC)- and RAS-transformed mouse fibroblasts, and that the transformed morphology of these cells can be reversed by expressing the transactivation domain deletion mutant of c-Jun (TAM67). Here, we found that lysyl oxidase (Lox), encoding an extracellular matrix-modifying enzyme, is downregulated in a c-Jun-dependent manner in ODC-transformed fibroblasts (Odc cells). In addition to Lox, the Lox family members Lox-like 1 and 3 (Loxl1 and Loxl3) were found to be downregulated in Odc as well as in RAS-transformed fibroblasts (E4), whereas Lox-like 4 (Loxl4) was upregulated in Odc and downregulated in E4 cells compared to normal N1 fibroblasts. Tetracycline-regulatable LOX re-expression in Odc cells led to inhibition of cell growth and invasion in three-dimensional Matrigel in an activity-independent manner. On the contrary, LOX and especially LOXL2, LOXL3, and LOXL4 were found to be upregulated in several human melanoma cell lines, and LOX inhibitor B-aminopropionitrile inhibited the invasive growth of these cells particularly when co-cultured with fibroblasts in Matrigel. Knocking down the expression of LOX and especially LOXL2 in melanoma cells almost completely abrogated the invasive growth capability. Further, LOXL2 was significantly upregulated in clinical human primary melanomas compared to benign nevi, and high expression of LOXL2 in primary melanomas was associated with formation of metastases and shorter survival of patients. Thus, our studies reveal that inactive pro-LOX (together with Lox propeptide) functions as a tumor suppressor in ODC- and RAS-transformed murine fibroblasts by inhibiting cell growth and invasion, and active LOX and LOXL2 as tumor promoters in human melanoma cells by promoting their invasive growth.

Identification of integrins alpha6 and beta7 as c-Jun- and transformation-relevant genes in highly invasive fibrosarcoma cells.

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c-Jun- and transformation-related gene expression changes in S-adenosylmethionine decarboxylase (AdoMetDC)-overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline-inducible dominant-negative mutant of c-Jun (TAM67) or c-Jun shRNA. Among the small set of target genes detected were integrins alpha6 and beta7, cathepsin L and thymosin beta4, all upregulated in the AdoMetDC-transformed cells and downregulated upon reversal of transformation by TAM67 or c-Jun shRNA. The upregulation of integrin alpha6 subunit, pairing with integrin beta1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin alpha6 or beta1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC-transformants and human HT-1080 fibrosarcoma cells in three-dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin alpha6 staining in high-grade human fibrosarcomas. Our data show that c-Jun can regulate all three key steps of invasion: cell adhesion (integrin alpha6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin beta4). In addition, this is the first study to associate integrin beta7, known as a leukocyte-specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin alpha6beta1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin alpha6beta1, alone or combined with inhibitors of cathepsin L and thymosin beta4, as chemotherapeutic agents.

Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.

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.

Reversible regulation of the transformed phenotype of ornithine decarboxylase- and ras-overexpressing cells by dominant-negative mutants of c-Jun.

c-Jun is an oncogenic transcription factor involved in the regulation of cell proliferation, apoptosis and transformation. We have previously reported that cell transformations induced by ornithine decarboxylase (ODC) and c-Ha-ras oncogene, commonly activated in various cancer cells, are associated with constitutively increased phosphorylation of c-Jun on Ser residues 63 and 73. In the present study, we examined the significance of c-Jun phosphorylation and activation on the phenotype of the ODC- and ras-transformants, by using specific inhibitors and dominant-negative (DN) mutants to c-Jun NH(2)-terminal kinase (JNK) and its upstream kinase, SEK1/MKK4 (mitogen-activated protein kinase kinase 4), and to c-Jun. The transformed morphology of both the ODC- and ras-expressing cells was reversed partially by JNK inhibitors and DN JNK1, more effectively by DN SEK1/MKK4 and phosphorylation-deficient c-Jun mutants (c-Jun(S63,73A), c-Jun(S63,73A,T91,93A)) and most potently by a transactivation domain deletion mutant of c-Jun (TAM67). Moreover, tetracycline-inducible TAM67 expression in ODC- and ras-transformed cells showed that the transformed phenotype of the cells is reversibly regulatable. TAM67 also inhibited the tumorigenicity of the cells in nude mice. These inducible cell lines, together with their parental cell lines, provide good models to identify the genes and proteins relevant to cellular transformation.