Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance.

Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation.

Evaluation of novel cationic gene based liposomes with cyclodextrin prepared by thin film hydration and microfluidic systems.

In gene delivery, non-viral vectors have become the preferred carrier system for DNA delivery. They can overcome major viral issues such as immunogenicity and mutagenicity. Cationic lipid-mediated gene transfer is one of the most commonly used non-viral vectors, which have been shown to be a safe and effective carrier. However, their use in gene delivery often exhibits low transfection efficiency and stability. The aim of this study was to examine the effectiveness of novel non-viral gene delivery systems. This study has investigated the encapsulation and transfection efficiency of cationic liposomes prepared from DOTAP and carboxymethyl-ß-cyclodextrin (CD). The encapsulation efficiency of the CD-lipoplex complexes were also studied with and without the addition of Pluronic-F127, using both microfluidic and thin film hydration methods. In vitro transfection efficiencies of these complexes were determined in COS7 and SH-SY5Y cell lines. Formulation stability was evaluated using liposomes size, zeta potential and polydispersity index. In addition, the external morphology was studied using transmission electron microcopy (TEM). Results revealed that formulations produced by microfluidic method had smaller, more uniform and homogenious size and zeta-potential as well as higher encapsulation efficiency when compared with liposomes manufactured by thin film hydration method. Overall, the results of this study show that carboxymethyl-ß-cyclodextrin increased lipoplexes' encapsulation efficiency using both NanoAssemblr and rotary evaporator manufacturing processes. However, this increase was reduced slightly following the addition of Pluronic-F127. The addition of carboxymethyl-ß-cyclodextrin to cationic liposomes resulted in an increase in transfection efficiency in mammalian cell lines. However, this increase appeared to be cell line specific, COS7 showed higher transfection efficiency compared to SH-SY5Y.

Insights into the Influences of Carboxymethyl-ß-Cyclodextrin on DNA Formulations Characteristics and Gene Transfection Efficiency.

BACKGROUND: Gene therapy is an expanding field and it can treat genetic and acquired diseases. OBJECTIVE: It was found that formulations with DNA: CM-ß-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-ß-CD are the most stable formulations indicating high incorporation of DNA within CM-ß -CD. METHOD: Gel electrophoresis revealed DNA with low CM-ß -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-ß-CD and 1:3:3 DNA: CM-ß-CD: Pluronic-127 show no DNA fragment, suggesting good condensation of DNA inside cyclodextrin cavity. RESULTS: This was confirmed by fluorescence data where fluorescence intensity was reduced for samples DNA: CM-ß-CD 1:3. Overall, the findings showed that Carboxymethyl-beta-cyclodextrin (as a novel non-viral gene vector) was able to provide condensation and protection to the DNA, with and without Pluronic-F127, at low concentration. CONCLUSION: pDNA/CM-ß-CD complex has not only shown to be able to transfect COS 7 and SHSY5Y cell lines, but it gives a higher transfection efficiency than that produced by the TransIT-LT1 commercial transfection reagent.

Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis.

BACKGROUND AND AIMS: Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. RESULTS: Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. CONCLUSIONS: We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma.

Stem Cell Markers in Neuroblastoma-An Emerging Role for LGR5.

The prognostic value of cancer stem cell markers in various cancer subtypes is a well documented research area. Our findings show that the stem cell marker Lgr5 is associated with an aggressive phenotype in neuroblastoma. Here, we discuss these findings within the context of recent studies in several cancers such as lung, colorectal and intestinal cancer, glioblastoma and ewing's sarcoma. Neuroblastoma continues to be an elusive disease, due to its heterogeneous presentation ranging from spontaneous regression to aggressive metastatic disease and intertwined genetic variability. Currently, the most significant prognostic marker of high risk disease and poor prognosis is amplification of the MYCN oncogene, which is found in approximately 25% of cases (Huang and Weiss, 2013). With this in mind, there is still much to learn about the driving mechanisms of this aggressive pediatric tumor. Neuroblastoma development is thought to be the result of aberrant differentiation of the cell of origin, embryonic neural crest cells which then migrate and invade during the developmental stage (Joshi et al., 2007). Aberrant cells are those which would, under normal conditions form the mature tissues of the sympathetic ganglia and adrenal medulla. Tumors are known to develop indiscriminately along the radius of the sympathetic ganglia, although it is well established that the adrenal glands are fundamentally the most common primary site (Jessen and Mirsky, 2005).

A novel model of urinary tract differentiation, tissue regeneration, and disease: reprogramming human prostate and bladder cells into induced pluripotent stem cells.

BACKGROUND: Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation. OBJECTIVE: To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation. DESIGN, SETTING, AND PARTICIPANTS: Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used. RESULTS AND LIMITATIONS: Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies. CONCLUSIONS: IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression.

BACKGROUND: MYCN oncogene amplification occurs in 20-25% of neuroblastoma and is associated with a poor prognosis. We previously reported that MYCN amplified (MNA) p53 wild-type neuroblastoma cell lines failed to G1 arrest in response to irradiation, but this could not be attributed to MYCN alone. HYPOTHESIS: Genes co-amplified with MYCN and/or the predominant cell type, neuronal (N) or substrate adherent (S) phenotypes determine the downstream response to DNA damage in neuroblastoma cell lines. METHODS: The MYCN amplicons of five MNA and two non-MNA cell line were mapped using 50K Single Nucleotide Polymorphism (SNP) arrays. One MNA (NBL-W) and one non-MNA neuroblastoma cell line (SKNSH) were sub-cloned into N and S-type cells and the p53 pathway investigated after irradiation induced DNA damage. To determine the role of p53 it was knocked down using siRNA. RESULTS: No genes with a potential role in cell cycle regulation were consistently co-amplified in the MNA cell lines studied. High MYCN expressing NBLW-N cells failed to G1 arrest following irradiation and showed impaired induction of p21 and MDM2, whereas low MYCN expressing NBLW-S cells underwent a G1 arrest with induction of p21 and MDM2. Conversely N type cells underwent higher levels of apoptosis than S type cells. Following p53 knockdown in SHSY5Y N-type cells there was a decrease in apoptosis. CONCLUSIONS: The downstream response to DNA damage in p53 wild-type neuroblastoma cell lines is p53 dependent, and determined both by the morphological sub-type and MYCN expression.

High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma.

PURPOSE: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14(ARF) pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. HYPOTHESIS: Inactivation of the p53/MDM2/p14(ARF) pathway develops during treatment and contributes to neuroblastoma relapse. METHODS: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14(ARF) methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization. RESULTS: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14(ARF) inactivation in 12 of 41 (29%) cases: 3 had p14(ARF) methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. CONCLUSIONS: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated.