engleski

Methylation patterns of DKK1, DKK3, SFRP4, and GSK3β are accompanied with different expression levels in human astrocytoma

We investigated genetic and epigenetic changes and protein expression levels of negative regulators of Wnt signaling, DKK1, DKK3, SFRP4 and APC as well as GSK3β and β-catenin in 64 human astrocytomas of grades II–IV. Methylation-specific PCR revealed promoter methylation of DKK1, DKK3, SFRP4 and GSK3β in 38%, 43%, 16% and 18% of samples, respectively. Grade IV comprised the lowest number of methylated GSK3β cases and highest of DKK3. Methylation of SFRP4 was present exclusively in grade II astrocytomas (72.8%) and appeared significantly more often than in grade III (p=0.022) and grade IV (p<0.001). Evaluation of the immunostaining using H-score was performed for β-catenin, both total and unphosphorylated (active) forms. Additionally, active (pY216) and inactive (pS9) forms of GSK3β protein were analyzed. Prevalence of β-catenin’s active form (rs=0.634, p<0.001) was confirmed in astrocytoma tumor cells. We revealed that astrocytoma with higher levels of the active pGSK3β-Y216 had lower expression levels of its inactive form (p<0.0001, Z=-5.332). Changes in APC’s exon 11 were observed in 44.44% of samples by PCR/RFLP. Astrocytomas with changes of APC had higher H-score values of total β-catenin compared to the group without genetic changes (t=-2.264, p=0.038). Furthermore, a positive correlation between samples with methylated DKK3 promoter and the expression of active pGSK3β-Y216 (rs=0.356, p=0.011) was established. Our results emphasize the importance of methylation for the regulation of Wnt signaling. Large deletions of APC associated with increased β-catenin levels, together with oncogenic effects of both β-catenin and GSK3β, are clearly involved in astrocytoma evolution. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations.

astrocytic brain tumors ; Wnt signaling ; DKKs ; SFRP4, GSK3β ; APC ; β-catenin

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