Novel Signaling Pathways and Therapeutic Targets in Neuroblastoma

Neuroblastoma is the most common solid tumor in children under the age of five. Unfortunately, it is also a childhood cancer that carries a very poor prognosis.

Neuroblastoma originates from embryonic neural crest cells. Tumors typically arise from the adrenal gland, but can also develop in sympathetic nervous tissues in other parts of the body. Because it is a highly heterogeneous cancer, neuroblastoma is difficult to treat. Patients often relapse, and those who do are left with few effective treatment options. Children who are considered high-risk patients have survival rates as low as 40%.^1,2

Only modest improvements in survival rates for children with neuroblastoma have been reported in the last 20 years. Technological advances, however, now enable us to stratify patients according to their disease “risk,” meaning we can treat those with a greater chance of relapse more aggressively from the outset. MYCN is a well-known example of an oncogene used to identify children who are likely to experience more aggressive disease; however, the way in which MYCN affects prognosis is unknown.³

Despite advances in genomics we do not understand many of the pathways involved in neuroblastoma. What we do know is that tyrosine kinases play an important role in neuroblastoma tumorigenesis. DeNardo et al. (2013) recently identified a unique phosphoproteomics approach to target neuroblastoma cell lines. By looking at phosphorylation, they were able to identify multiple receptors and downstream mediators. Targeting these together may yield better therapeutic outcomes for young cancer patients.⁴

The investigators used an NB10 cell line, which has increased MYCN expression and is resistant to chemotherapy. They also used a human neural precursor cell line (NPC) to model a normal baseline neural crest cell. Using shotgun phosphoproteomics they were able to sequence a collection of phosphopeptides, which they sequenced using liquid chromatography–mass spectrometry.

To determine the differences in phosphopeptide abundance in neuroblastoma cells compared with normal neural crest derived cells, researchers isolated proteins from the NB10 cell line, representing a high-risk MYCN amplified cell line, and from an NPC line, representing a control human neural crest cell line. They then ran two experiments in parallel. For the total phosphoproteome, titanium dioxide enrichment was used to isolate the phosphopeptides. To achieve the more sensitive detection of the tyrosine phosphoproteome, the research team used immune-affinity precipitation with a phosphotyrosine antibody.

DeNardo et al. performed mass spectrometry studies using a linear trap quadrupole LTQ Orbitrap Velos mass spectrometer (Thermo Scientific), and the spectra were collected in positive ion mode. The resulting spectra were then searched against the non-redundant, human Uniprot complete proteome set database.

Quantitative data were calculated automatically on selected ion chromatogram peak areas for every assigned peptide. The scientists used the resulting data to construct a heat map, in order to perform a quantitative comparison of phosphopeptides in NB10 and NPC cells.

The authors identified 2,598 unique serene, threonine and tyrosine phosphopeptide sequences in both cell lines using a 1% false discovery rate; 2,181 unique phosphorylation sites were identified on 1,171 proteins. Serine phosphorylation represented 1,768 sites, followed in abundance by threonine phosphorylation and then by 174 tyrosine phosphorylation sites. They also found 176 phosphorylation sites on 151 protein kinases. A significant proportion of the phosphorylation sites that DeNardo et al. identified had not previously been reported in neuroblastoma.

In order to gain an understanding of the biological processes and molecular functions of the phosphopeptides they identified, the research team categorized them by biological processes and molecular function. In doing so, they implicated several intracellular signaling pathways in neuroblastoma biology. They showed that MYCN neuroblastoma cells contain an overabundance of phosphopeptides, resulting from phosphorylation of tyrosine kinases such as IGF-1R/IR and Ret, which are known drivers of growth and metastasis in neuroblastoma and other cancers. The activation of IGF-1R/IR and Ret appeared to upregulate PI3K/Akt/mTor and Raf/MEK/ERK pathways.

This study by DeNardo et al. is the first quantitative phosphoproteomic analysis of a neuroblastoma cell line, demonstrating that it is possible to quantify signaling networks within a cancer cell and to correlate this with functional information. It shows a potentially new way to develop treatments for neuroblastoma that are potentially more effective because multiple receptors and pathways may be targeted simultaneously.

 

References

1. American Cancer Society. (2013) “5-year survival rates for neuroblastoma based on risk groups,” available at http://www.cancer.org/cancer/neuroblastoma/detailedguide/neuroblastoma-survival-rates.

2. Albanus, R.D.A., et al. (2013) “Reverse Engineering the Neuroblastoma Regulatory Network Uncovers MAX as One of the Master Regulators of Tumor Progression,” PLoSONE, doi: 10.1371/journal.pone.0082457.

3. Weinstein, J.L., Katzenstein, H.M., and Cohn, S.L. (2003) “Advances in the Diagnosis and Treatment of Neuroblastoma,” The Oncologist, 8(3) (pp. 278–92).

4. DeNardo, B.D., et al. (2013) “Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signalling Pathways in Neuroblastoma,” PLoSONE, doi: 10.1371/journal.pone.0082513.

Post Author: Miriam Pollak. Miriam specialised in neuroscience as an undergraduate but traded in lab work for a post graduate degree in science communication.

She has since had a career that has spanned science communication, science education and communications management.

However, Miriam has found her bliss balancing her love of writing and disseminating medical research with managing a multimillion dollar research budget for a childhood cancer charity in Australia.