Hyaluronan activates cell motility of v-Src-transformed cells via Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase-Akt in a tumor-specific manner.

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src-transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44-neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src-transformed cells.

Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ovarian cancer cells.

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have shown previously that treatment of ovarian cancer cells with peritoneal conditioned medium or purified fibronectin (FN) activated matrix metalloproteinase 9 secretion and, thereby, cancer cell invasion. By use of antisense oligonucleotides to focal adhesion kinase (FAK) and a dominant-negative mutant of ras (S17Nras), we found that both FAK and c-Ras were required for the activation of matrix metalloproteinase 9 secretion by FN. In addition, both antisense oligonucleotides to FAK and S17Nras inhibited mitogen-activated protein kinase activation by FN treatment, suggesting the involvement of mitogen-activated protein kinase in the FN-dependent signaling.

The Ras-mitogen-activated protein kinase pathway is critical for the activation of matrix metalloproteinase secretion and the invasiveness in v-crk-transformed 3Y1.

To search for the intracellular signaling pathway critical for the secretion of matrix metalloproteinases (MMP), we studied the effects of dominant negative Ras (S17N Ras) and dominant negative MEK1 (MEK1AA) expression in v-crk-transformed 3Y1. Expression of either S17N Ras or MEK1AA dramatically suppressed the augmented secretion of MMP-2 and MMP-9 in v-crk-transfected 3Y1. Similarly, a Ras farnesyltransferase inhibitor, manumycin A, and a MEK1 inhibitor, U0126, suppressed MMP secretion in a dose-dependent manner, whereas a PI3 kinase inhibitor, wortmannin, could not. In addition, the suppression of MMP secretion by S17N Ras showed good correlation with the inhibition of in vitro invasiveness of the cells. In contrast, expression of dominant negative C3G did not suppress MMP secretion, although it substantially blocked the c-Jun N-terminal kinase activation. Taken together, the Ras-MEK1 pathway, but not the C3G-JNK pathway, seems to play a key role in the activation of MMP secretion and, hence, the invasiveness of v-crk-transformed cells.

Molecular cloning and characterization of human MAWD, a novel protein containing WD-40 repeats frequently overexpressed in breast cancer.

A full-length cDNA clone encoding a novel protein containing WD-40 repeats, which were frequently involved in protein-protein interactions, was isolated and sequenced. This clone had a predicted open reading frame (ORF) encoding 350 amino acids possessing six repeats of WD-40 motif. It was most closely homologous to TRIP-1, a phosphorylation substrate of the transforming growth factor-beta type II receptor. In the process of characterizing the function of the new gene product, we found that overexpression of the gene seemed to activate mitogen-activated protein kinase and to promote anchorage-independent growth of the cells. Moreover, the gene product was frequently overexpressed in human tumor breast tissues compared with their normal breast tissues, suggesting that the gene might be involved in the tumor progression. Radiation hybrid mapping placed the gene into human chromosome 12q11-12 near the marker D12S1593.

Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of src.

To search for the biochemical properties of cells relevant to transformation by p60v-src, we examined the activities and amounts of metalloproteinase (gelatinase) released from chicken embryonic fibroblasts infected with various mutants of Rous sarcoma virus by zymography and immunoblotting. While nontransforming Src proteins, including cellular p60c-src and its nonmyristylated form, had no stimulatory effect, wild-type p60v-src and the transforming mutant of cellular p60c-src stimulated the secretion and proteolytic activation of metalloproteinases. Moreover, the activation of metalloproteinase secretion strongly correlated with the invasiveness of cells assayed by the modified Boyden Chamber method. Chimeric mutants between v-src and c-src, which are transforming but produce less distinct morphological changes in infected cells, also stimulated the secretion of metalloproteinases as well as wild-type p60v-src. Deletion mutants of v-Src in which varying portions of the NH2-terminal half of p60v-src are deleted stimulated secretion to a level similar to that of wild-type regardless of the degree of morphological change they induce. Together with Src protein, other oncogene products including Yes, Fps, ErbB and Crk were also found to stimulate the secretion of metalloproteinases. Thus, these results suggest that transformation of cells with src and other oncogenes is closely associated with the enhanced secretion of metalloproteinases that may play an important role in tumor invasion and metastasis.

Suppression of cell spreading by v-Crk requires Ras-MEK-MAP kinase signaling.

We investigated the attachment and spreading of v-Crk-transformed cells, v-Crk3Y1, on fibronectin. Transformation by v-Crk virtually suppressed the spreading, but not the attachment, of cells on fibronectin. This suppression of cell spreading was not correlated with the suppression of integrin alpha5 and beta1 expression. However, the spreading of v-Crk3Y1 on fibronectin was dramatically restored by either expression of dominant-negative Ras or treatment with manumycin A, a Ras farnesyltransferase inhibitor. Moreover, both expression of dominant-negative MEK1 and treatment of cells with U0126, a MEK1 inhibitor, restored the cell spreading of v-Crk3Y1. In contrast, neither treatment with LY294002, a PI3K inhibitor, nor expression of dominant-negative C3G showed no effect on cell spreading on fibronectin. Taken together, our results suggest that, among multiple signaling pathways activated by v-Crk, the Ras-MEK1-MAP kinase cascade plays a pivotal role in the suppression of cell spreading on fibronectin, but C3G and the PI3 kinase do not.

A role for FAK in the Concanavalin A-dependent secretion of matrix metalloproteinase-2 and -9.

To study the signaling pathway critical for the secretion of matrix metalloproteinases (MMPs), we examined the role of focal adhesion kinase (FAK) in Concanavalin A (Con A)-stimulated cells. We established a cell line in which FAK gene was conditionally inducible by use of FAK-null fibroblasts and the tetracycline repression system. In this cell line, FAK expression was undetectable in the presence of tetracycline but induced within 1 day by the removal of the drug. We found that FAK expression augmented the Con A-dependent secretion of MMP-9 and MMP-2. In contrast, proteolytic activation of MMP-2 by Con A-treatment did not require FAK expression. In addition, activation of MMP-secretion and tyrosine phosphorylation of FAK by Con A, but not the proteolytic activation of MMP-2, required attachment of the cells to the extracellular matrix. Taken together, our results suggest that the FAK signaling pathway play a pivotal role in the secretion of MMPs.

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

Ras pathway is required for the activation of MMP-2 secretion and for the invasion of src-transformed 3Y1.

To search for the signaling pathway critical for tumor invasion, we examined the effects of dominant negative ras (S17N ras) expression on the activation of matrix metalloproteinase-2 (MMP-2) in src-transformed 3Y1, SR3Y1, under the control of conditionally inducible promoter. In SR3Y1 clones transfected with S17N ras, augmented secretion and proteolytic activation of MMP-2 were dramatically suppressed by S17N Ras expression, while tyrosine phosphorylation of cellular proteins was not suppressed. We found that invasiveness of SR3Y1 cells assayed by the modified Boyden Chamber method was strongly suppressed by S17N Ras expression. In contrast, cell morphology reverted partially and glucose uptake remained unchanged by S17N Ras expression. In addition, treatment of SR3Y1 with manumycin A, a potent inhibitor of Ras farnesyltransferase, strongly suppressed both augmented secretion and proteolytic activation of MMP-2. Contrary, treatment of SR3Y1 with wortmannin or TPA showed no clear effect on MMP-2 activation. Thus, these results strongly suggest that Ras-signaling, but neither P13 kinase- nor protein kinase C-signalings, plays a critical role in activation of MMP-2 and, subsequently, in the invasiveness of src-transformed cells.

c-Ras is required for the activation of the matrix metalloproteinases by concanavalin A in 3Y1 cells.

Concanavalin A (Con A) is known to trigger augmented secretion and proteolytic activation of the matrix metalloproteinases (MMPs) in fibroblasts. To study the signaling pathway critical for the activation of MMPs in fibroblasts, we examined the effects of dominant negative ras (S17N ras) expression under the control of conditionally inducible promoter in Con A-activated 3Y1 cells. We found that augmented secretion and proteolytic activation of MMP-2 and MMP-9 together with expression of MT1-MMP in Con A-activated 3Y1 were dramatically suppressed by S17N ras expression. These results strongly suggest that c-Ras plays a critical role in the augmented expression and proteolytic activation of MMPs in fibroblasts.

Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells.

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, P1-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the P13-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEKI-MAPK and P13K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer.

Suppression of cell growth by ectopic expression of N-cadherin.

We found that ectopic expression of N-cadherin in 3Y1 caused tight association of cells and, thereby, substantially suppressed cell growth. N-cadherin expression inhibited neither tyrosine phosphorylation of cellular proteins, GTP uptake onto Ras, nor activation of MAP kinase, suggesting that it does not directly interfere the Ras-MAP kinase pathway. However, co-expression of N-cadherin with dominant negative Ras, S17N Ras, showed synergestic growth inhibitory effect, suggesting that N-cadherin signaling antagonizes the Ras-MAP kinase signaling. In addition, we found that N-cadherin yielded cell-cycle arrest at G0/G1 phase. These results strongly suggest that N-cadherin cell adhesion machinery works as a negative controller of cell cycle in 3Y1 and this growth suppressive function of cadherin is distinct from the epithelial morphogenetic function.

Hyaluronan activates mitogen-activated protein kinase via Ras-signaling pathway.

Hyaluronan (HA) triggers a wide variety of cellular functions, yet its signaling pathway remains largely unclear. We found that HA-treatment of 3Y1 cells activated tyrosine phosphorylation of cellular proteins and mitogen-activated protein (MAP) kinase in a time- and dose-dependent manner, and, subsequently, stimulated cell growth. This HA-activity was resistant to boiling at 100 degrees C but completely abolished by treatment with hyaluronidase, suggesting that HA itself, but not any HA-associated proteins, has the activity. In addition, we found that HA-dependent activation of MAP kinase was strongly suppressed by the expression of dominant negative ras (S17N ras). These results suggest that Ras-MAP kinase pathway is activated by HA and may play an important role in HA-dependent signaling.

Constitutive activation of MAP kinase kinase (MEK1) is critical and sufficient for the activation of MMP-2.

We investigated the role of MEK1 signaling in MMP-2 activation by use of constitutive active/dominant negative forms of MEK1 and MEK1-specific inhibitor. We found that cell transformation with active forms of MEK1 dramatically increased secretion and proteolytic activation of MMP-2 and subsequently stimulated invasiveness of cells. Contrary, expression of dominant negative form of MEK1 in v-src-transformed cells or in Con A-activated cells resulted in the suppression of the augmented secretion and proteolytic activation of MMP-2. In addition, treatment of v-src-transformed cells with PD98059, a MEK1-specific inhibitor, strongly suppressed the secretion and activation of MMP-2, whereas treatment with wortmannin, a PI3 kinase inhibitor, showed no clear effect on MMP-2 secretion. Taken together, these results strongly suggest that MEK-MAP kinase signaling, but not PI3 kinase signaling, plays a critical role in the activation of MMP-2 secretion and, subsequently, in the invasiveness of v-src-transformed cells.