Supplementary Figures 1-4 from Neuroblastoma-derived secretory protein is a novel secreted factor overexpressed in neuroblastoma
ARTICLE ABSTRACT
Secreted proteins such as growth factors, cytokines, and chemokines play important roles in tumor development. Through expression microarray and bioinformatic analysis, we discovered a novel secreted protein, neuroblastoma-derived secretory protein (NDSP). The NDSP gene is found on chromosome 1q25.2 and encodes a 167 amino acid protein with a putative signal peptide. Using real-time PCR and immunoblotting, we find that NDSP is specifically overexpressed in neuroblastoma at much higher levels than other adult and pediatric malignancies and normal tissues. NDSP is an 18-kDa protein that can be secreted by NDSP-transfected HEK-293T cells, as well as, neuroblastoma cell lines endogenously expressing NDSP. Inhibiting NDSP expression in neuroblastoma cell lines with retrovirally transduced NDSP small hairpin interfering RNA, sh-NDSP, results in decreased cellular proliferation and colony formation. We also find inhibited extracellular signal-regulated kinase (ERK)1/2 phosphorylation in the sh-NDSP cell line. Treating the parental cell line with MAP/ERK kinase 1/2 inhibitors, which diminish ERK1/2 phosphorylation, results in decreased cell proliferation. Culturing these transduced cells with recombinant NDSP, reintroducing NDSP overexpression in the knockdown cell line, or inducing Ras oncogene overexpression for constitutive ERK1/2 activation results in a reversal of the growth-inhibited phenotype and proliferation rates similar to the control cells. In addition, reintroduction of NDSP overexpression in the sh-NDSP cell line results in ERK1/2 phosphorylation similar to control. We conclude that NDSP is specifically overexpressed in neuroblastoma and actively secreted from tumor cells. Furthermore, NDSP serves as a growth factor for neuroblastoma tumor cells through activation of the ERK-mediated proliferation pathway. [Mol Cancer Ther 2009;8(8):2478–89]
