Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12

Abstract

Activated forms of Ras family members are prevalent in many cancers where Ras mutants transduce signals essential for transformation, angiogenesis, invasion and metastasis. As a cancer progression model, we used NIH3T3 cells to explore the mechanism of Ras-induced tumorigenesis. Ras family mutants H-RasV12 and Rit79L strongly induced foci formation, while Rho family mutants RhoA-QL, Rac1-QL and Cdc42-QL were less effective. A comparison of downstream transcriptional targets of Ras and Rho family members using a 26 383 element cDNA microarray revealed that the osteopontin (OPN) gene exhibited the best correlation between magnitude of gene expression change and level of foci formation (r ¼ 0.96, Po0.001). In association with H-RasV12-and Rit79L-mediated transformation, foci secreted OPN protein and upregulated the OPN receptor CD44, suggesting the novel initiation of an aberrant OPN-CD44-Rac autocrine pathway. In support of this were the following observations. First, RGD-deficient OPN protein-binding activity was present in H-RasV12-transformed cells but not in control cells, and binding activity was inhibited by the CD44 blocking antibody. Second, foci formation, cell invasion and Rac activity were induced by H-RasV12 and inhibited by the CD44 blocking antibody. Third, foci formation by H-RasV12 was substantially reduced by a short interfering RNA (siRNA) specifically targeting OPN expression for knockdown. Fourth, H-RasV12-mediated transformation was not blocked by the GRGDS peptide, suggesting that OPN effects were not mediated by the integrins. Lastly, OPN knockdown affected the downstream expression of 160 '2nd tier' genes, and at least a subset of these genes appears to be involved in transformation. Indeed, four genes were selected for knockdown, each resulting in a disruption of foci formation and/or invasion. These results underscore the role of aberrant autocrine signaling and transcriptional networking during tumorigenesis.

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.…”

“…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).…”

“…Rac signaling is also implicated in invasive and metastatic cell growth (Kurisu et al, 2005), and it was recently suggested that autocrine activation of an OPN-CD44-Rac pathway in Ras-transformed cells enhances transformation and motility (Teramoto et al, 2005). Based on the observation that ectopic expression of rac2 leads to agar colony formation in CEF (cf.…”

The multi-functional cellular adhesion molecule CD44 is regulated by the 8;21 chromosomal translocation

“…In addition, we observed the deregulation of osteopontin (OPN1) a ligand of CD44 17 and the Runx2 transcription factor that positively regulates OPN1. 18 The increased expression of these factors suggest a feedback loop for continued expression of CD44, 39 signaling that is favorable for transformation 49,50 and leukemic stem cell maintenance (Figure 4e). Our finding that AML1-ETO9a and AML1-ETO bind and regulate the CD44 promoter resulting in increased CD44 expression in a murine progenitor line and in primary AML1-ETO9a murine leukemia stem cells suggests (1) that AML1-ETO9a/AML1-ETO regulation of CD44 expression and function on leukemia initiating stem cells in the periphery and in the bone marrow niche may be one of the early events in t(8;21) leukemogenesis and (2) that this direct regulation of a CD44 network, which includes OPN and Runx2, promotes a continued signaling pathway that is essential in survival.…”

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome

“…Addressing CD44 as a functional receptor for OPN would be important to explore the molecular mechanism underlying dysplastic changes induced by the OPN/CD44 axis. It is known that CD44 triggering stimulates diverse signalling pathways, including activation of ERK (Bourguignon et al, 2005), RAC (Teramoto et al, 2005), and RHO (Bourguignon et al, 2003), as well as secretion of soluble factors, like cytokines and metalloproteinases (Zhang et al, 2002;Bourguignon et al, 2003;Murphy et al, 2005). These pathways are potentially involved in dysplastic changes induced by OPN/CD44v6.…”