Olga Piskareva

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

Background Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD2), providing a target for tumor-specific delivery. Principal Findings Nanoparticles encapsulating miR-34a and conjugated to a GD2 antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. Significance These novel findings highlight the potential of anti-GD2-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD2-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Modulation of neuroblastoma disease pathogenesis by an extensive network of epigenetically regulated microRNAs

MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3′-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.

The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high‐risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high‐risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non‐MYCN amplified tumour cells, an SK‐N‐AS subline (SK‐N‐AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse‐selection process. High resolution aCGH analysis of SK‐N‐AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK‐N‐AsCis24 and in parental SK‐N‐AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR‐520f, was also demonstrated to be partially responsible for increased NAIP levels in SK‐N‐AsCis24. Interestingly, miR‐520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug‐resistance and have implications for future therapeutic research.

MicroRNA-497 impairs the growth of chemoresistant neuroblastoma cells by targeting cell cycle, survival and vascular permeability genes.

Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy.

Assessment of Basic Biological Functions Exerted by miRNAs

Assessment of cell viability and proliferation under different miRNA expression levels is an important step in the evaluation of basic miRNA functional effects within the cell. Here, we describe the overexpression of miRNA in question in cells achieved by transfection with subsequent examination of cell viability and proliferation over a period of time using the acid phosphatase assay.

Endogenous exosome labelling with an amphiphilic NIR-fluorescent probe

The recognition of the biological, diagnostic and medical importance of exosomes has given rise to an urgent need for efficient labelling of these extracellular vesicles in ways that do not alter their inherent characteristics. We report for the first time an endogenous method to NIR-fluorescent labelled exosomes using an amphiphilic probe without the need for immunolabelling or synthetic or chromatographic manipulation of exosomes. Comparative analyses of labelled and unlabelled exosomes with NTA, AFM, flow cytometry and immunoblot analysis all show a high degree of similarity. Spectroscopic analysis and fluorescence imaging confirmed the ability to visualise purified NIR-exosomes.

Chapter 13 – Neuroblastoma

Neuroblastoma is a highly malignant pediatric cancer accounting for approximately 15% of all childhood cancer deaths. There is strong experimental evidence that the genome-wide methylation and miRNA expression play a critical role in neuroblastoma pathogenesis. The purpose of the chapter is to review growing evidence of epigenetic changes contributing to aggressive disease development along with the future and currently available therapeutics.