8828

NRP2

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-beta1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-beta1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFbeta-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFbeta-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-beta1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

The epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET), are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGF-beta1 and ZEB1-induced EMT. Here, we report that neuropilin (NRP)-2, the high-affinity receptor for SEMA3F and a coreceptor for certain growth factors, is upregulated during TGF-beta1-driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TbetaRI dependent and SMAD independent, occurring mainly at a posttranscriptional level involving increased association of mRNA with polyribosomes. Extracellular signal-regulated kinase (ERK) and AKT inhibition blocked NRP2 upregulation, whereas RNA interference-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. In addition, NRP2 attenuation inhibited TGF-beta1-driven morphologic transformation, migration/invasion, ERK activation, growth suppression, and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGF-beta1-mediated growth inhibition. In support of these results, human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGF-beta1-induced EMT in lung cancer.