f ADAM17-silenced tumors. Discussion ADAM17 promotes MC38CEA tumor development in the absence of EGFR and ErbB4 signaling ADAM17 is a predominant sheddase for a number of growth factors of EGF family including TGFa, HB-EGF and amphiregulin. In many tumors overexpression of these growth factors and their receptors is accompanied by a high expression of ADAM17. The availability of growth factors ADAM17 in Tumor Development in their soluble form is usually required for efficient receptor activation thus the shedding of growth factors is viewed as a major event responsible for ADAM17 pro-tumorigenic effects. We show that MC38CEA tumors derived from ADAM17-silenced cells develop slower than tumors from control cells. Surprisingly, this difference cannot be attributed to the impaired shedding of ligands for EGFR or ErbB4, whose role in tumorigenesis is well established, because MC38CEA cells appear to lack these receptors. Does ADAM17 contribute to MC38CEA tumor development through NRG-1 shedding The fact that MC38CEA cells express NRG-1 and both subunits of its heterodimeric receptor compelled 9 ADAM17 in Tumor Development Serum TNF IFNc MCP-1 6.2115.81 1.011.74 60.5120.09 Tumor lysates 5.7334.03 7.6717.34 769.973164.45 The values represent the lowest average and the highest average cytokine concentrations estimated in ADAM17-silenced tumor lysates and in sera from mice bearing ADAM17-silenced tumor taken from three independent experiment. Concentrations of cytokines in the sera of control healthy mice: TNF 5.061.4; IFNc 
1.560.5; MCP-1 20.664.6. doi:10.1371/journal.pone.0050791.t001 us to consider the possibility that ADAM17-dependent protumorigenic phenotype of MC38CEA cells at least partially depends on NRG-1 ErbB2/ErbB3 axis. Despite the fact that ErbB2 is ligandless and ErbB3 lacks kinase activity, the ErbB2/ ErbB3 heterodimer is considered the most potent signaling receptor of the family in respect to cell proliferation and transformation. Many of NRG-1 isoforms are subjected to shedding. Some studies point to ADAM17 as the major 15976016 NRG-1 sheddase but other recognize ADAM9, ADAM19, and BACE1 as 25137254 the key enzymes for NRG-1 shedding. Even though MC38CEA express ADAM9 and ADAM19 as evaluated by RT-PCR, ADAM17 must be a dominant NRG-1 sheddase in these cells as its,80% silencing resulted in 10 ADAM17 in Tumor Development ,60% inhibition of NRG-1 release and decreased phosphorylation of ErbB2. It remains unclear why NRG-1 did not stimulate proliferation of MC38CEA cells. The consequences of NRG-1 binding to ErbB2/ErbB3 heterodimer may vary depending on the cell type, the set and the ratio of the EGF receptor family members expressed on the cell surface, the expression pattern of intracellular signaling molecules, and probably a number of extracellular factors. It must be emphasized that NRG-1 isoforms, apart from their ability to MedChemExpress 871700-17-3 switch on cell proliferation under certain conditions, are crucial for directing differentiation during organogenesis. The situation in which the pro-tumorigenic potential of ADAM17-expressing cells is not correlated with increased cell proliferation, is not limited to MC38CEA colon carcinoma. We observed that silencing of ADAM17 had no effect on the proliferation rate of B16F10 melanoma cells but strongly inhibited tumor growth after s.c. injection of the cells into syngeneic mice. Similarly, silencing of ADAM17 expression decreased the invasiveness of pancreatic ductal adenocarcinoma cells as tested in vitro in matrige
