The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This alternative pathway of osteoclast activation involves a nonadhesive interaction between neuroblastoma cells and bone marrow mesenchymal stem cells. Stimulated bone marrow mesenchymal stem cells express markedly increased levels of interleukin-6, which is then responsible for osteoclast activation. This report describes a critical role of bone marrow mesenchymal stem cells in bone destruction in cancer. (Cancer Res 2005; 65(4): 1129-35)

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.

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 ®broblasts 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. Oncogene (2000) 19, 5539 ± 5542.Keywords: matrix metalloproteinase; MMP-2; MMP-9; FAK; Concanavalin A; signal transduction One of the critical steps for tumor invasion is the destruction of extracellular matrix catalyzed by matrix metalloproteinases (MMPs) (Liotta, 1986). MMPs such as MMP-2 and MMP-9 are a family of neutral proteinases, secreted from the cells as inactive zymogens, require Zn or Ca ions for their activity and proteolytic cleavage is essential for their activation (Stetler-Stevenson et al., 1989). In human cancer tissues, augmented secretion and proteolytic activation of MMPs often associated with tumor metastasis (Kataoka et al., 1996). However, the signaling pathways critical for MMP secretion remain largely unclear.To study the signaling pathway critical for MMP secretion, we have examined their activities in cells stimulated with Concanavalin A (Con A) (Thant et al., 1997), ®bronectin (Shibata et al., 1997(Shibata et al., , 1998 as well as cells transformed with oncogenes (Hamaguchi et al., 1995;Thant et al., 1999) as a model. In these studies, the Ras-MEK1-MAPK signaling played a critical role in MMP secretion, whereas anti-sense experiment suggested that focal adhesion kinase (FAK) was required for the enhanced MMP-9 secretion in FNstimulated ovarian cancer cells (Shibata et al., 1998). FAK is localized to focal adhesion contacts, activated by cell attachment to the extracellular matrix and appears to regulate cell attachment and motility (Clark and Brugge, 1995;Ilic et al., 1995), so that it was somewhat unexpected to ®nd the involvement of FAK in MMP-9 secretion.To con®rm and extend our previous observations, we studied the role of FAK in the Con A-dependent secretion of MMP by use of a conditionally inducible system for FAK together with FAK-null ®broblasts. Con A, a tetravalent lectin, is known to trigger augmented secretion and proteolytic activation of MMPs in ®broblasts, hence widely used for the study of MMP secretion (Hurum et al., 1982; Overall and Sodek, 1990). Here, we show that expression of FAK in FAK gene-targeted cells recovers t...

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