Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines

Abstract

A metastatic phenotype can be induced in benign rat mammary cells (Rama 37 cells) by transfecting them with metastasis-inducing DNAs (Met-DNAs). Stable transfection of Met-DNAs increases the level of the metastasis-associated protein, osteopontin. Randomly picked clonal cell lines have been established from the pool of Rama 37 cells transfected with one metastasis-inducing DNA, C9-Met-DNA. In these cell lines, moderate correlation is observed between the copy number of C9-Met-DNA and their metastatic potential (linear regression coefficient, R 2 ¼ 0.48). A very close correlation is observed between the cell lines' metastatic potential in vivo and the osteopontin mRNA levels in vitro (R 2 ¼ 0.74), but not with another metastasis-associated protein in this system, S100A4 (R 2 ¼ 0.21). A close correlation is also observed between osteopontin mRNA levels and the adhesive potential (R 2 ¼ 0.91) of the cells, but not with their growth rate in vitro (R 2 ¼ 0.03). These observations support the previous suggestion that osteopontin is the direct effector of C9-Met-DNA and that the presence of C9-Met-DNA is necessary, if not sufficient, for the induction of metastasis in vivo in this system. Additionally, these results suggest that Rama 37 cells with increased osteopontin mRNA levels become metastatic not through an increased growth rate, but through an increase in cellular adhesiveness.

Cooperative cell transformation by Myc/Mil(Raf) involves induction of AP-1 and activation of genes implicated in cell motility and metastasis

“…Figure 5d), demonstrating intrinsic oncogenic potential of this gene in a direct cell transformation assay. This extends previous results showing that this gene promotes metastatic potential when introduced into established rat mammary epithelial cell lines (Moye et al, 2004;Teramoto et al, 2005). The transforming potential of OPN is enhanced by concomitant ectopic expression of the 126MRP gene that is also activated in v-myc/v-mil-transformed cells, suggesting that OPN and 126MRP are able to act in a synergistic manner.…”

“…Intriguingly, OPN and members of the S100 and Rho gene families have been implicated in molecular processes linked to cell migration, invasion, and metastasis (Marenholz et al, 2004;Rittling and Chambers, 2004;Kurisu et al, 2005). We show that transcriptional activation of OPN, a typical marker gene for metastatic tumor cells (Denhardt et al, 2001;Weber, 2001;Moye et al, 2004;Rittling and Chambers, 2004), is dependent on cFos which is highly expressed in v-myc/v-mil(raf)-transformed cells. Furthermore, ectopic expression of OPN, 126MRP, or Rac2 proteins from a retroviral vector causes partial transformation of primary avian fibroblasts, suggesting that these gene products are critically involved in oncogenesis induced by Myc/ Mil(Raf), possibly in a joint signaling pathway.…”

“…Originally discovered as a phosphoprotein secreted by transformed cells in culture (Senger et al, 1979), it is now recognized as a multifunctional protein implicated in development, neoplastic change, tumor progression and metastasis, but is also abundant in the mineral matrix of bones binding tightly to hydroxyl apatite (Mi et al, 2004;Rittling and Chambers, 2004). OPN not only represents a classical marker for metastatic cells, but has proven intrinsic potential to enhance the metastatic phenotype of tumor cells (Moye et al, 2004;Rittling and Chambers, 2004;Teramoto et al, 2005). Although there is strong evidence that overexpression of OPN at the mRNA and protein level represents a key molecular event in tumor progression and metastasis, the molecular mechanisms and signaling pathways leading to its transcriptional activation and its precise biochemical function in these disease-related processes are largely unknown (Mi et al, 2004;Rittling and Chambers, 2004).…”

“…The cytokine OPN identified here as a putative target of v-myc/v-mil signaling has been implicated in cell transformation and metastasis, and is regarded as a candidate target for cancer therapy (Weber, 2001;Moye et al, 2004;Rittling and Chambers, 2004). Hence, we decided to study the regulation of the avian OPN gene Figure 1d), primary chicken embryo fibroblasts (CEF) were freshly transformed by the MH2 virus (CEF/MH2) and passaged.…”

“…To facilitate detection of the protein products by using FLAG antibodies , the FLAG epitope was inserted between the start methionine and the second amino-acid residue of each protein (Figure 5a). Since the human homologs of both OPN and 126MRP have been implicated in tumorigenesis (Donato, 1999;Denhardt et al, 2001;Marenholz et al, 2004;Moye et al, 2004), bicistronic retroviral constructs for the simultaneous overexpression of OPN and 126MRP, and also of OPN and rac2, were engineered to test for possible synergistic effects in cell transformation. Protein expression in transfected and metabolically labeled cells was monitored by immunoprecipitation using antibodies directed against the FLAG peptide or against a 220-amino-acid recombinant OPN protein (Figure 5b).…”