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Role of the MET/HGF receptor in proliferation and invasive behavior of osteosarcoma¹

N. COLTELLA^*,2, M. C. MANARA, V. CERISANO, L. TRUSOLINO, M. F. DI RENZO^*, K. SCOTLANDI and R. FERRACINI^*

Laboratory of
^* Cancer Genetics and
Division of Molecular Oncology of the Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, Candiolo, Italy; and
Oncologic Research Laboratory, Istituti Ortopedici Rizzoli, Bologna, Italy

²Correspondence: Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, Km 3.95, 10060 Candiolo (To), Italy. E-mail: nadia.coltella{at}ircc.it

SPECIFIC AIMS

Signal transduction downstream HGF receptor (MET) activation involves multiple pathways that account for mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. We have analyzed the effect of MET receptor activation in five human osteosarcoma cell lines, evaluating levels of HGF-dependent activation of MAPK and PKB/AKT as biochemical readouts of mitogenic and invasive responses, respectively.

PRINCIPAL FINDINGS

1. Characterization of the active pathways downstream of MET activation in human osteosarcoma cell lines
We examined the signaling pathways relevant to cell proliferation and motility activated by HGF in five human osteosarcoma cell lines (U-2OS, MG-63, Saos-2, IOR/OS9, IOR/OS10). We studied the MAP kinase cascade that transduces signals from active Ras protein, modulating cell growth and proliferation. As shown in Fig. 1 A ,HGF stimulation induced ERK1/2 phosphorylation in the following cell lines: U-2OS, Saos-2, IOR/OS9, and IOR/OS10. The phosphorylated active ERK1/2 forms translocated to the nuclear cellular fraction and showed an increased enzymatic activity toward physiological substrates. Phosphorylation and activation were rapid, becoming strongly detectable at the earliest point analyzed, then decreased at subsequent times. We also studied the PI3K/AKT pathway involved in signaling motility and protection from apoptosis mediated by the MET receptor. Activation of PI3K pathway results in phosphorylation and consequently activation of serine/threonine kinase PKB/AKT. In two cell lines (U-2OS, Saos-2) HGF induced a consistent phosphorylation of PKB/AKT (Fig. 1B ), whereas a modest response was detected in MG-63 and IOR/OS10 cells.

View larger version (67K): [in this window] [in a new window]   Figure 1. MAPK and PKB/AKT activation by HGF in osteosarcoma cell lines. Osteosarcoma cell lines were starved for 24 h in medium devoid of serum, then stimulated with HGF at 40 ng/mL for the indicated times. Total lysates were resolved by SDS-PAGE, Western blotted, and immunoprobed with antibodies against MAPK (A panels) or PKB/AKT (B). For each cell line, total proteins were labeled with antibodies directed against either the active form (upper panel) or total protein (lower panel) The active forms of both kinases were identified with antibodies specific for the phosphorylated forms, where phospho-residues are the regulatory ones. The anti-active MAPK antibody detects the dually phosphorylated, active form of the mitogen-activated protein kinase (MAPK) enzymes (ERK1 and ERK2), as it binds the phosphorylated residues corresponding to the Thr 183 and Tyr 185 of the mammalian ERK2 enzyme. PKB/AKT activation was estimated as phosphorylation in Western blot analysis of total cell extracts with anti-phospho AKT polyclonal antibodies directed against the Ser 473 of AKT1. These antibodies also recognize the equivalent serine residues in AKT2 and AKT3. The presence of equal amounts of AKT protein was estimated by labeling blots with anti-AKT protein antibodies.

2. HGF induces proliferation in MET expressing human osteosarcoma cells
To demonstrate the HGF-dependent proliferative response in osteosarcoma cell lines, cell cultures were incubated with HGF (100 ng/mL). Cell growth was evaluated after 2 days of treatment. HGF stimulated growth in the absence of serum of all but one osteosarcoma cell line. In MG-63, where the MAPK cascade was not activated by MET, HGF alone was not enough to stimulate cell growth, as expected. HGF-dependent growth was consistently inhibited by the MAPK-specific inhibitor PD98059 and by a MET kinase inhibitor (K252a). This showed that cell lines lacking the MAPK response to HGF do not proliferate in response to HGF (Fig. 2 ).

View larger version (30K): [in this window] [in a new window]   Figure 2. Schematic diagram. HGF stimulates the MAPK and the PI3K signal transduction pathways in osteosarcoma cells leading to proliferation and motility/invasiveness. MAPK and PI3-kinase cooperation emerging from the analysis of different osteosarcoma cell lines representative of different high-grade osteosarcomas suggests how the HGF/SF and MET receptor couple might function in activating biological properties that might contribute to osteosarcoma progression.

3. HGF induces osteosarcoma cell motility and invasiveness
Cell lines displaying HGF-induced activation of PKB/AKT—namely, U-2OS and Saos-2—showed an ability to migrate through the micropore filter of a Transwell chamber in response to HGF (Table 1 ). The migration induced by HGF was completely reverted by the MET kinase inhibitor K252a. As expected, other serum factors induced a motile response in U-2OS and Saos-2. Both cell lines presented a basal motility that was doubled by serum alone. This response is independent from HGF, as it was not inhibited by K252a. PKB/AKT activation plays a central role in motility of these cells. In fact, serum- and HGF-stimulated motility was significantly inhibited by low concentrations (10 µM) of the specific PI3K inhibitor LY294002 in the presence of 10% FCS.

4. HGF induces osteosarcoma cell invasion of collagen matrix
We evaluated whether osteosarcoma cells are able to invade 3-dimensional collagen gels after HGF stimulation, which in most epithelial cell lines leads to the formation of branched cords and invasion. HGF alone induced spreading and branching of U-2OS cells starting from a concentration of 2 ng/mL, whereas fetal bovine serum did not. Concentrations >10 ng/mL HGF increased dramatically the length and number of branched cords.

CONCLUSIONS AND SIGNIFICANCE

HGF input might be important to osteosarcoma genesis as 1) MET receptors are expressed at a high level in almost 100% of human osteosarcomas; 2) HGF is ubiquitous but mainly produced by mesenchymal cells and tumors; 3) mesenchymal cell transformation has been attained with expression of MET receptor in cells secreting the factor or by aberrantly expressing a MET kinase activated by either translocation or mutations.

Here we show that HGF stimulates MAPK and the PI3K signal transduction pathways in osteosarcoma cells leading to proliferation and motility/invasiveness. In response to HGF, osteosarcoma cells proliferate and invade depending on their ability to activate either of the two pathways, respectively (Fig. 2) .

HGF stimulation of epithelial and nonepithelial cell lines such as skin keratinocytes, melanocytes, kidney tubular cells, and hepatocytes induces cell growth. Here we show that a proliferative response to HGF is associated with a cell’s ability to show MAPK cascade activation by HGF in osteosarcoma cells.

Human osteosarcomas show rapid growth in vivo and in culture. We have analyzed the properties of a series of cell lines, all derived from high-grade osteosarcomas, that commonly show an invasive/metastatic phenotype. Metastasis and invasion are distinguishing features of osteosarcoma, frequently a disseminated disease, as 80% of primary osteosarcomas have associated macro- and micrometastases at presentation. Musculoskeletal tumors other than osteosarcomas or rhabdomyosarcomas expressed minor amounts of the MET receptor in a significant percentage of cases. These included nonossifying fibromas, osteoblastomas, desmoplastic fibromas of bone, chondroblastomas, and giant cell tumors of bone. More than 50% of these tumors scoring positive for MET expression were associated with recurrent or locally aggressive lesions. The MET receptor may be relevant to osteosarcoma progression, as its activation by HGF confers functions for metastatic diffusion and local and peripheral invasion of the surrounding tissues. Metastasis is a multistep process: motility and invasion of basement membrane represent key steps in the process. We found that HGF stimulates osteosarcoma cell motility in cell lines where MET activation also induced AKT activation. We also show that osteosarcoma cells are able to invade collagen matrix in an HGF-dependent way, providing the cells can show an AKT response to HGF.

In conclusion, the picture of MAPK and PI3-kinase cooperation emerging from analysis of different osteosarcoma cell lines representative of different high-grade osteosarcoma suggests how the HGF/SF and MET receptors might function together in activating biological properties that may contribute to osteosarcoma progression. The effectiveness of a biological inhibitor such as K252a in modulating kinase activity and the biological response to HGF in vitro might hold therapeutic promise as a drug in treating osteosarcoma patients.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0576fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0576fje

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