c-Kit is preferentially expressed in MYCN-amplified neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571

Coexpression for c-Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c-Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c-Kit was found in 10/75 and for SCF in 17/75, with 5/10 c-Kit-positive tumors also expressing SCF. For both, c-Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c-Kit was detected in 8/14 amplified vs. 2/61 single copy (p<0.001), and SCF in 9/14 amplified vs. 8/61 single copy tumors (p<0.001). Furthermore, the association of c-Kit expression with advanced stage (3 or 4) (p=0.001) and of SCF expression with adrenal primary (p=0.03) was substantiated. The in vitro activity of the tyrosine kinase inhibitor STI-S71 (imatinib mesylate, Gleevec, Glivec) on NB cell lines positive or negative for c-Kit was also assessed. When cells were grown in 10% fetal calf serum, the 4 c-Kit-positive cell lines tested were sensitive to STI-571 growth inhibition to a different extent (ranging from 30 to 80%); also the c-Kit-negative cell line GI-CA-N was slightly affected, suggesting that other STI-571 targets operate in regulating NB proliferation. In addition, c-Kit-positive cell lines SK-N-BE2(c) and HTLA230, grown in SCF only, remained sensitive (40 and 70% of growth inhibition, respectively), while, in the same conditions, proliferation of the c-Kit-negative cell line GI-CA-N was not affected. Immunoprecipitation of c-Kit from cell lysates of SK-N-BE2(c) and HTLA230 cells grown in SCF and subsequent western blot analysis of the immunoprecipitates revealed a sharp decrease of c-Kit phosphorylation after STI-571 treatment. These data demonstrate that both c-Kit and SCF are preferentially expressed in vivo in the most aggressive neuroblastic tumors and that their signaling is active in promoting in vitro NB cell proliferation that can be selectively inhibited by treatment with STI-571. (C) 2003 Wiley-Liss, Inc.