Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines

Abstract

Abstract. Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of 'stemness genes' such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition, glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.

Mechanisms involved in selecting and maintaining neuroblastoma cancer stem cell populations, and perspectives for therapeutic targeting

“…CD133 and c-kit are considered to be markers of NB CSCs, CSC survival, growth regulation and poor clinical outcome[ 12 , 100 ]; GPRC5C is considered to indicate neuroectodermal origin and a differentiated embryonic stem cell state; Notch is involved in neural SC maintenance[ 101 - 103 ]; Pigf2 is involved in tumour angiogenesis growth and survival[ 104 ], and neurotrophin receptors are involved in NB growth, invasion and survival[ 16 , 100 ]. Other reverse transcription-polymerase chain reaction studies have reported increased CD133, Sox2, Bmi1 and Msi1 expression in immature NB CSC-like cells[ 105 ], and genome-wide microarray analysis of CSCs enriched from NB cell lines have identified Wnt, Notch, Shh and TGFβ signalling pathways in self-renewing CSC-like phenotypes, with TGFβ activation associated with repression of SMAD6 and SMAD7 TGFβ signalling repressors, Shh signalling associated with expression of Patched, Gli1 and smoothened, and CD133 and CD15 expression enriched in CSC-like neurospheres[ 106 ].…”

“…The mRNA-binding protein Musashi1 is a critical regulator of SC self-renewal and has been implicated in CSC-like self-renewal capacity in NB by promoting target mRNA translation and stem cell-fate transition[ 105 , 221 ]. Although the signalling mechanisms involved in Musashi1 function in stem/progenitor cells are unclear, Musashi1 is regulated by phosphorylation and by MAPK signalling in oocytes, and exhibits functional duality by promoting translation and repressing mRNA targets, in a similar way to Musashi2, which represses TGFβR1 mRNA, whilst activating translation of SMAD3 mRNA in proliferating K562 myelogenous leukemia cells[ 222 ].…”

Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer

“…CD133, a penta-span, tri-membrane glycoprotein encoded by Prominin-1 was originally found to be expressed in human hematopoietic stem and progenitor cells. It is also reported to be highly expressed in several cancers, including GBM and NB, making the CD133 + CSC more metastatic and resistant to radiotherapy as well as chemotherapy [7,8,9]. CD133 + CSC possess the same functional properties such as the ability to differentiate and self-renew as their normal counterpart, thus contributing toward tumor initiation and proliferation.…”