Introduction: Neuropilins (NRP) are membrane receptors capable of binding two disparate ligands, vascular endothelial growth factors (VEGF) and class 3 semaphorins (SEMA), and regulating two diverse systems, angiogenesis and neuronal guidance. It has been reported that most tumor cells express NRP1 while VEGFR1 and 2 are less commonly expressed. Our studies on rhabdomyosarcoma (RMS) showed that NRP1 is expressed in several tumor cell lines, however, the functional activities including tumor growth and angiogenesis remain unclear. Hence, here we investigated the role of NPR1 over expression in RMS. Methods: 7 RMS cell lines (RD, Rh6, A204, Rh4, Rh18, Rh28, and Rh30) were analyzed for VEGF and VEGF receptor expression by qPCR and western blot analysis. Results: All 7 subtypes of VEGF and NRP1 were found to be highly expressed by q PCR and western blot analysis. Rh30 and A204 cells were selected for further NRP1 study since they express low Flt1 and no KDR or VEGFR3. We generated stable transfectant clones expressing NRP1 in Rh30 and A204 cells. These clones showed more than 5 times higher NRP1 expression than their vector control. Interestingly, the proliferation of NRP1 over-expressing clones in Rh30 was significantly reduced compared to the vector control. MTT assay showed a 32% reduction (n =4; p = 0.0022) in proliferation while Trypan Blue analysis showed a 47% decrease in cell number (p=0.0113). Furthermore, clonogenic assays showed significant decrease of colony growth of NRP1 over-expressing clones. We also report a similar degree of reduction in proliferation and growth in A204 cell derived NRP1 over-expressing clones. We hypothesized that NRP1 over expression may inhibit VEGF mediated proliferative signaling. To investigate this possibility, we performed qPCR analysis and found that NRP1 over expression was associated with up-regulation of all 7 VEGF subtypes and all VEGF receptors including Flt1 sFlt1, sNRP1 sNRP2, KDR and VEGFR3 while signal pathway genes AKT, ERK, JUN, JNK and mTOR were dramatically down regulated. Since the MAPK pathway is associated with cell proliferation and survival, down regulation of these genes might explain the reduction in RMS cell proliferation. Hence, we performed MAPK/ERK1,2 phosphorylation studies and found that MAPK/ERK1,2 phosphorylation was induced in Rh30 and A204 cells following treatment with recombinant human VEGF165 administration. This phosphorylation was not affected by siRNA Flt1 knockdown. Conclusion: Our results indicate that NRP1 may reduce cell proliferation and growth in RMS by down regulating MAPK/ERK1,2 signaling. This mechanism may be relevant in other non-mesenchymal tumors as well.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA