Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors.
It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.
Shahi
Mehdi H
MH
Brain Tumor Biology Unit-CIFA, University of Navarra School of Sciences, Pamplona, Spain.
Schiapparelli
Paula
P
Afzal
Mohammad
M
Sinha
Subrata
S
Rey
Juan A
JA
Castresana
Javier S
JS
D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D044127:Epigenesis, Genetic; D053823:Hedgehog Proteins / Q000235:genetics* / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000071676:Zinc Finger Protein GLI1
D004273:DNA, Neoplasm; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; C000715631:NKX2-2 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; C509239:SHH protein, human; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1