Involvement of aldehyde dehydrogenase 1A2 in the regulation of cancer stem cell properties in neuroblastoma.

Despite the introduction of 13-cis-retinoic acid (13-cis-RA) into the current chemotherapy, more than half of high-risk neuroblastoma patients have experienced tumor relapses driven by chemoresistant cancer stem cells (CSCs) that can be isolated by their ability to grow as spheres. Although aldehyde dehydrogenase (ALDH) has been used to characterize CSCs in certain cancers, ALDH remains elusive in neuroblastoma. In the present study, we determined ALDH activity and expression of its 19 isoforms in spheres and parental cells of neuroblastoma. ALDH activity and several ALDH isoforms were consistently induced in spheres of different neuroblastoma cells. While ALDH1A2, ALDH1L1 and ALDH3B2 expression was consistently induced in spheres and associated with the sphere and colony formation, only ALDH1A2 expression was significantly correlated with the poor prognosis of neuroblastoma patients. ALDH1A2 expression was further associated with the growth and undifferentiation of neuroblastoma xenografts and the resistance of neuroblastoma cells to 13-cis-RA. These results suggest that ALDH1A2 is involved in the regulation of CSC properties in neuroblastoma.

Minimal residual disease monitoring in neuroblastoma patients based on the expression of a set of real-time RT-PCR markers in tumor-initiating cells.

Minimal residual disease (MRD) is derived from tumor-initiating cells (TICs) and is responsible for tumor relapse. Neuroblastoma is characterized by extreme tumor heterogeneity, and more than half of high-risk patients experience tumor relapse. To overcome tumor heterogeneity and achieve more sensitive detection of MRD, several sets of real-time RT-PCR markers have been reported for MRD monitoring in neuroblastoma patients from different centers. However, these markers vary across centers and are still being validated. In the present study, we validated the ability of 14 commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11 real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range. By using this protocol, we prospectively monitored MRD in 73 bone marrow (BM), 12 peripheral blood stem cell and 8 peripheral blood samples from 14 neuroblastoma patients treated at a single center. We scored 100, 56, 56 and 57% BM cytology-positive, elevated vanillylmandelic acid (VMA), elevated homovanillic acid (HVA) and elevated neuron-specific enolase (NSE) samples as MRD-positive, respectively. MRD was also positive in 48, 45, 46 and 43% of the BM cytology-negative and normal VMA, normal HVA and normal NSE samples, respectively. These results suggest that the present MRD detection protocol based on the expression of a set of 11 real-time RT-PCR markers in neuroblastoma TICs achieves sensitive MRD monitoring in neuroblastoma patients.

Rab15 expression correlates with retinoic acid-induced differentiation of neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer deaths. Although retinoic acid (RA) is currently used to treat high-risk neuroblastoma patients in the clinic, RA-responsiveness is variable and unpredictable. Since no alterations in the RA-signaling pathway have been found in neuroblastoma cells, molecules correlated with RA-induced differentiation will provide predictive markers of RA-responsiveness for clinical use. The Rab family of small G proteins are key regulators of membrane traffic and play a critical role in cell differentiation and cancer progression. Although an increasing number of cancer-associated alternative splicing events have been identified, alternative splicing of Rab proteins remains to be characterized in neuroblastoma. In the present study, we focused on Rab15 that was originally identified as a brain-specific Rab protein and regulates the endocytic recycling pathway. We identified alternatively spliced Rab15 isoforms designated as Rab15CN and Rab15AN in neuroblastoma cells. Rab15CN was composed of 7 exons encoding 212 amino acids and showed brain-specific expression. Alternative splicing of exon 4 generated Rab15AN that was predicted to encode 208 amino acids and was predominantly expressed in testis. RA induced neuronal differentiation of neuroblastoma BE(2)-C cells and specifically up-regulated Rab15CN expression. Reciprocally, RA-induced differentiation was observed in Rab15CN-expressing BE(2)-C cells in preference to Rab15AN-expressing BE(2)-C cells. Furthermore, Rab15CN expression was also specifically up-regulated during RA-induced differentiation of newly established neuroblastoma cells from high-risk patients. These results suggest that Rab15 expression correlates with RA-induced differentiation of neuroblastoma cells.