Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma: the oncogenic role of the ligands.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. METHODS: The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. RESULTS: The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. CONCLUSIONS: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

miRNA-7 and miRNA-324-5p regulate alpha9-Integrin expression and exert anti-oncogenic effects in rhabdomyosarcoma.

The prognosis of patients with metastatic rhabdomyosarcoma (RMS), the most common type of soft tissue sarcoma in children, is poor and no strategies have been identified to improve their dismal prognosis. Alpha-9 integrin (ITGA9) plays a particularly crucial role in cancer progression and invasiveness. Despite the consensus on the remarkable pro-oncogenic potential of this protein, the miRNA-mediated regulation of ITGA9 has barely been studied to date. In the present study, miR-7 and miR-324-5p were selected as the best candidates after a screening to find ITGA9 regulators, and their effects on cell proliferation and invasion in RMS are described and characterized for the first time. Interestingly, the overexpression of both miRNA produced a clear impairment of cell proliferation, while miR-7 also induced a remarkable drop in cell invasion. Furthermore, the stable overexpression of both miRNA was found to reduce tumor growth in orthotopic RMS models and miR-7 was able to impair metastatic lung colonization. Consequently, we conclude that miR-7 and miR-324-5p show anti-oncogenic and anti-metastatic potential, thereby opening up the possibility of being used as novel therapeutic tools to avoid RMS progression.

Patient-derived xenografts for childhood solid tumors: a valuable tool to test new drugs and personalize treatments.

The use of preclinical models is essential in translational cancer research and especially important in pediatric cancer given the low incidence of each particular type of cancer. Cell line cultures have led to significant advances in cancer biology. However, cell lines have adapted to growth in artificial culture conditions, thereby undergoing genetic and phenotypic changes which may hinder the translational application. Tumor grafts developed in mice from patient tumor tissues, generally known as patient-derived xenografts (PDXs), are interesting alternative approaches to reproducing the biology of the original tumor. This review is focused on highlighting the interest of PDX models in pediatric cancer research and supporting strategies of personalized medicine. This review provides: (1) a description of the background of PDX in cancer, (2) the particular case of PDX in pediatric cancer, (3) how PDX can improve personalized medicine strategies, (4) new methods to increase engraftment, and, finally, (5) concluding remarks.

SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150.

SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis.

BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways.

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Mu-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase.

Opioid peptides and alkaloids exert their effects via G protein-coupled receptors (GPCRs). It has been shown that, in addition to trophic factors, some GPCRs are able to activate the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) signal transduction pathway, thus leading to cell survival. The aim of this study was to test whether activation of mu-opioid receptors has protective effects on serum withdrawal-induced cell death and to study the possible implication of PI 3-K in this process. In SH-SY5Y neuroblastoma cells fully differentiated by exposure to retinoic acid for five days, the enkephalin derivative selective mu-agonist DAMGO (0.1-2 microM) and the alkaloid morphine (0.1-10 microM) promoted cell survival after serum deprivation (MTT and trypan blue exclusion assays), without inducing cell proliferation. These effects were fully reversed by naloxone, by the selective mu-antagonist beta-funaltrexamine (beta-FNA) and also by the specific PI 3-K inhibitor LY294002. The two agonists stimulated Akt phosphorylation and the effect was also abolished by beta-FNA and by LY294002. In mouse primary cortical neurons, DAMGO reduced the percentage of apoptosis after 6, 12, 24 and 48 h of serum withdrawal; as determined by Hoechst staining. This effect was blocked by beta-FNA, by pre-treatment with pertussis toxin and by LY294002. DAMGO also stimulated Akt phosphorylation via PI 3-K in this primary neuronal culture. Together, these results indicate that stimulation of the mu-opioid receptor promotes neuronal survival in a G(i/o)-linked, PI 3-K-dependent signaling cascade and suggest that Akt may be a key downstream kinase involved in this anti-apoptotic effect.

MYCN repression of Lifeguard/FAIM2 enhances neuroblastoma aggressiveness.

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

Efficient in vivo microRNA targeting of liver metastasis.

Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors.

Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate.

Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.