Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.     

AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

Cat-1/Git-1 is a multifunctional protein that acts as a GTPase-activating protein (GAP) for Arf GTPases, as well as serves as a scaffold for a number of different signaling proteins. Cat-1 is best known for its role in regulating cell shape and promoting cell migration. However, whether Cat-1 might also contribute to cellular transformation is currently unknown. Here we show that ～95% of cervical tumor samples examined overexpress Cat-1, suggesting that the up-regulation of Cat-1 expression is a frequent occurrence in this type of cancer. We demonstrate further that knocking down Cat-1 from NIH3T3 fibroblasts expressing an activated form of Cdc42 (Cdc42 F28L), or from the human cervical carcinoma (HeLa) cell line, inhibits the ability of these cells to form colonies in soft agar, an in vitro measure of tumorgenicity. The requirement for Cat-1 when assaying the anchorage-independent growth of transformed fibroblasts and HeLa cells is dependent on its ability to bind paxillin, while being negatively impacted by its Arf-GAP activity. Moreover, the co-expression of Cat-1 and an activated form of Arf6 in fibroblasts was sufficient to induce their transformation. These findings highlight novel roles for Cat-1 and its interactions with the Arf GTPases and paxillin in oncogenic transformation.     

Complete restoration of cell surface activity of transmembrane-truncated MT1-MMP by a glycosylphosphatidylinositol anchor. Implications for MT1-MMP-mediated prommp2 activation and collagenolysis in three-dimensions

MT1-MMP is a potent collagenase not only required for skeletal development but also implicated in tumor invasion and metastasis. The mechanism through which cellsdeploy MT1-MMP to mediate collagenolysis remains largely unknown. C-terminally truncated MT1-MMP lacking its transmembrane and cytoplasmic domains, although proteolytic active in purified forms, is known to be deficient in cell-mediated proMMP2 activation and collagenolysis, suggesting that cells regulate its activity through both domains. Indeed, the cytoplasmic domain is recognized by the trafficking machinery that mediates its internalization and recycling. Here we demonstrate that its transmembrane domain can be functionally substituted by the glycosylphosphatidylinositol (GPI)-anchor of MT6-MMP. The GPI-anchored MT1-MMP, or MT1-GPI, activates proMMP2 on the cell surface and promotes cell growth in a three-dimensional type I collagen matrix. On the other hand, a GPI-anchored MMP13 with a functional furin activation signal fails to promote cell growth in a three-dimensional collagen matrix, whereas remaining competent in collagenolysis on a two-dimensional collagen matrix under serum-free conditions. alpha(2) macroglobulin (alpha(2)M) or serum is sufficient to inhibit the collagenase activity of GPI-anchored active MMP13. Our results suggest that both membrane-tethering and proteolytic activity encoded by MT1-MMP are required for its ability to promote cell growth and invasion in a three-dimensional collagen matrix.