Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth.

Neuroblastoma prognosis is dependent on both the differentiation state and stromal content of the tumor. Neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here, we identified critical growth-limiting components of the differentiating stroma secretome and designed a potential therapeutic strategy based on their central mechanism of action. We demonstrated that expression of heparan sulfate proteoglycans (HSPGs), including TßRIII, GPC1, GPC3, SDC3, and SDC4, is low in neuroblasts and high in the Schwannian stroma. Evaluation of neuroblastoma patient microarray data revealed an association between TGFBR3, GPC1, and SDC3 expression and improved prognosis. Treatment of neuroblastoma cell lines with soluble HSPGs promoted neuroblast differentiation via FGFR1 and ERK phosphorylation, leading to upregulation of the transcription factor inhibitor of DNA binding 1 (ID1). HSPGs also enhanced FGF2-dependent differentiation, and the anticoagulant heparin had a similar effect, leading to decreased neuroblast proliferation. Dissection of individual sulfation sites identified 2-O, 3-O-desulfated heparin (ODSH) as a differentiating agent, and treatment of orthotopic xenograft models with ODSH suppressed tumor growth and metastasis without anticoagulation. These studies support heparan sulfate signaling intermediates as prognostic and therapeutic neuroblastoma biomarkers and demonstrate that tumor stroma biology can inform the design of targeted molecular therapeutics.

Type III TGF-ß receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma.

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-ß receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TßRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TßRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TßRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TßRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TßRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.

The progesterone receptor hinge region regulates the kinetics of transcriptional responses through acetylation, phosphorylation, and nuclear retention.

Progesterone receptors (PRs) are critical regulators of mammary gland development and contributors to breast cancer progression. Posttranslational modifications of PR have been shown to alter hormone responsiveness. Site-directed mutagenesis demonstrated that upon hormone binding, PR is acetylated at the consensus sequence, KXKK (amino acids 638-641), located within the hinge region. We created an acetylation-deficient (K-A) mutant as well as acetylation mimics (K-Q or K-T). Interestingly, similar to K-A PR, PR acetylation mimics (K-Q or K-T) displayed delayed phosphorylation and nuclear entry relative to wild-type (wt) PR-B, indicative of disruption of PR nuclear-cytoplasmic shuttling. Wt PR-B, but not K-mutant PRs, induced c-myc at 1 h of progestin treatment. However, at 6 h of treatment, c-myc induction was comparable with levels induced by wt PR-B, suggesting that the precise timing of PR phosphorylation and nuclear retention are critical for cells to rapidly initiate robust transcriptional programs. In contrast to c-myc, progestin-induced serum- and glucocorticoid-regulated kinase (SGK) expression displayed sensitivity to PR acetylation but not nuclear entry. Namely, in the presence of progestin, acetylation-deficient (K-A) mutant PR-B up-regulated SGK mRNA relative to wt PR; progesterone response element-luciferase assays confirmed this result. However, K-Q and K-T acetylation mimics only weakly induced SGK expression independently of nuclear retention. These data reveal the ability of PR acetylation to alter the magnitude of transcriptional response at selected (slow response) promoters (SGK), whereas the hinge region dictates the kinetics of the transcriptional response to hormone at other (rapid response) promoters (c-myc). In sum, the PR hinge region is multifunctional. Understanding the ability of this region to couple acetylation, phosphorylation, and nuclear entry may provide clues to mechanisms of altered hormone responsiveness.