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Supplementary Figures 1-4 from Neuroblastoma-derived secretory protein is a novel secreted factor overexpressed in neuroblastoma

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posted on 2023-03-31, 23:22 authored by Sanjeev A. Vasudevan, Xiaoyi Shang, Shirong Chang, Ningling Ge, Jose L. Diaz-Miron, Heidi V. Russell, M. John Hicks, Andrew D. Ludwig, Catherine L. Wesson, Susan M. Burlingame, Eugene S. Kim, Javed Khan, Jianhua Yang, Jed G. Nuchtern
Supplementary Figures 1-4 from Neuroblastoma-derived secretory protein is a novel secreted factor overexpressed in neuroblastoma

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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]