Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma.

Approximately two-thirds of patients with neuroblastoma are found to have metastatic disease at time of diagnosis with frequent skeletal, lymph node, central nervous system, and liver involvement. Using a serial in vivo splenic injection model, we have isolated an aggressive subclone (BE(2)-C/LM2) from MYCN-amplified neuroblastomas that demonstrate an enhanced propensity to develop metastatic liver lesions. BE(2)-C/LM2 subclone cells demonstrate increased adherent, soft agar colony and tumorsphere growth in vitro. Components of the tumor microenvironment regulate cancer progression, via networks of cytokines and growth factors. Cytokine array analysis identified increased TIMP-1 in the plasma of mice injected with BE(2)-C/LM2 subclone cells, leading us to hypothesize that TIMP-1 may play a role in our observed prometastatic phenotype. Immunoblotting and ELISA demonstrated enhanced endogenous TIMP-1 expression in our isolated neuroblastoma subclone. Silencing endogenous TIMP-1 successfully blocked in vitro proliferation, soft agar colony formation and tumorsphere formation by BE(2)-C/LM2 cells. Stable RNA interference of endogenous TIMP-1 failed to reverse the prometastatic phenotype of our BE(2)-C/LM2 subclone in our liver metastasis model, suggesting that endogenous TIMP-1 levels may not be an essential component of this in vivo behavior. Notably, tissue microarray analysis and Kaplan-Meier by gene expression demonstrates that elevated TIMP-1 expression is correlated with increased disease relapse and mortality in patients with neuroblastoma. Taken together, our study identifies TIMP-1 as a novel soluble factor that is associated with a prometastatic phenotype in our in vivo model and adverse outcomes in patients with neuroblastoma.

Targeting gastrin-releasing peptide suppresses neuroblastoma progression via upregulation of PTEN signaling.

We have previously demonstrated the role of gastrin-releasing peptide (GRP) as an autocrine growth factor for neuroblastoma. Here, we report that GRP silencing regulates cell signaling involved in the invasion-metastasis cascade. Using a doxycycline inducible system, we demonstrate that GRP silencing decreased anchorage-independent growth, inhibited migration and neuroblastoma cell-mediated angiogenesis in vitro, and suppressed metastasis in vivo. Targeted inhibition of GRP decreased the mRNA levels of oncogenes responsible for neuroblastoma progression. We also identified PTEN/AKT signaling as a key mediator of the tumorigenic properties of GRP in neuroblastoma cells. Interestingly, PTEN overexpression decreased GRP-mediated migration and angiogenesis; a novel role for this, otherwise, understated tumor suppressor in neuroblastoma. Furthermore, activation of AKT (pAKT) positively correlated with neuroblastoma progression in an in vivo tumor-metastasis model. PTEN expression was slightly decreased in metastatic lesions. A similar phenomenon was observed in human neuroblastoma sections, where, early-stage localized tumors had a higher PTEN expression relative to pAKT; however, an inverse expression pattern was observed in liver lesions. Taken together, our results argue for a dual purpose of targeting GRP in neuroblastoma--1) decreasing expression of critical oncogenes involved in tumor progression, and 2) enhancing activation of tumor suppressor genes to treat aggressive, advanced-stage disease.

Gli1 transcriptional activity is negatively regulated by AKT2 in neuroblastoma.

Activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies including neuroblastoma. Expression of Gli1, a downstream effector of Hh, correlates with a favorable prognosis in patients with neuroblastoma. Moreover, Gli1 overexpression reduces mitotic index and induces transcription of genes involved in the differentiation of neuroblastoma cells; however, much remains unknown regarding the regulation of Gli1 transcriptional activity. Here, we report a novel negative regulation of Gli1 transcriptional activity by PI3K/AKT2 signal transduction pathway. Constitutively active PI3K subunit, p110α, inhibited Gli1 transcriptional activity in neuroblastoma cells, whereas, overexpression of an inactive form of PI3K subunit, p85, enhanced its activity. Specifically, the AKT2 isoform inhibited Gli1 luciferase activity. Silencing AKT2 using siRNA increased Gli1 transcriptional activity and conversely, overexpression of constitutively active AKT2 (myr-AKT2) decreased Gli1 transcriptional activity. Furthermore, Gli1 overexpression-mediated decrease in anchorage-independent growth was rescued by AKT2 overexpression. We also demonstrated that AKT2 overexpression regulates the nuclear-cytoplasmic distribution of exogenous Gli1 protein in neuroblastoma cells by relieving a GSK3ß-mediated destabilization of SUFU, a negative regulator of Gli1 nuclear translocation. Inhibition of nuclear Gli1 accumulation may explain for the suppression of the tumor-suppressive function of Gli1. Collectively, our findings suggest an important role of Gli1 as a tumor suppressor in neuroblastoma, and offer a mechanism by which AKT2 regulates the subcellular localization, and in turn, inhibits the tumor-suppressive function of Gli1 in neuroblastoma.