Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

Abstract

BackgroundThe Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors.MethodsWe silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.ResultsSilencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.ConclusionsOur results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

Nkx2-2as Suppression Contributes to the Pathogenesis of Sonic Hedgehog Medulloblastoma

“…However, we found that Nkx2-2as plays a tumor-suppressive role independent of Nkx2-2, consistent with the previous finding that in cis regulation of Nkx2-2 is dispensable for the prodifferentiation role of Nkx2-2as in neural stem cells (25). Furthermore, Shahi and colleagues found that Nkx2-2 is directly targeted and upregulated by Gli1 in MB cells (37), which in combination with our current findings suggests that this genomic region is involved in the pathogenesis of Shh-type MBs. While both transcription factors are critically involved in Shh signaling, previous studies suggested that Gli2 but not Gli1 is required for Shh signaling and mediates inappropriate activation of the pathway (38), which is in agreement with our finding that Nkx2-2as is downregulated solely by Gli2 via transcriptional activation of FoxD1.…”

Transcription Factor Targets as Treatment for Medulloblastoma

“…In general, Nkx2.2 expression was high in the low grade tumors (class II and III -anaplastic astrocytoma) compared to high grade glioblastoma multiforme (class IV-GBM) (Riemenschneider et al, 2004). Nkx2.2 is also expressed in medulloblastoma cell lines and primary tumor samples and Nkx2.2 expression is dependent on Shh-Gli1 pathway (Shahi et al, 2010). Upon knockdown of Gli1 in medulloblastoma cell line with short interfering RNAs (siRNA), Nkx2.2 expression was reduced compared to control negative siRNA treated cells (Shahi et al, 2010), suggesting that Gli1 positively regulates Nkx2.2 expression.…”

“…Interestingly, both Pax6 and Nkx2.2 are targets of the major Shh downstream transcription factor Gli1 in medulloblastoma cell lines and tissues (Shahi et al, 2010). Moreover, both genes have been implicated in stem cell maintenance (Hu et al, 2009;Zhang et al, 2010); therefore, it would be fruitful to understand these genes in the context of Shh signaling in TSC-mediated medulloblastoma biogenesis.…”

Targeting of sonic hedgehog-Gli signaling: A potential therapeutic target for patients with breast cancer

“…Bcl-2 and cyclin D1 are considered important target genes of the Shh signaling pathway, directly affecting cell proliferation and survival (33). Research indicates that high levels of cyclin D1 are associated with activation of the transcription factor Gli (34). Furthermore, the pro-survival protein Bcl-2 is upregulated in various tumors, including human breast cancers (35).…”