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Article

Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma

1
Laboratory of Neuro-Oncology, Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Šalata 12, 10 000 Zagreb, Croatia
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Department of Biology, School of Medicine, University of Zagreb, Šalata 3, 10 000 Zagreb, Croatia
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Department of Pathology, School of Medicine, University of Zagreb, Šalata 10, 10 000 Zagreb, Croatia
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Division of Pathology, University Hospital Center “Zagreb”, Kišpatićeva 12, 10 000 Zagreb, Croatia
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Department of Neurosurgery, University Hospital Center “Zagreb”, School of Medicine, University of Zagreb, Kišpatićeva 12, 10 000 Zagreb, Croatia
*
Author to whom correspondence should be addressed.
Academic Editors: Michael Kahn and Keane Lai
Cancers 2021, 13(11), 2530; https://doi.org/10.3390/cancers13112530
Received: 2 May 2021 / Revised: 17 May 2021 / Accepted: 19 May 2021 / Published: 21 May 2021
Astrocytomas are the most common type of primary brain tumor in adults. In this study, 64 astrocytoma samples of grades II–IV were analyzed for genetic and epigenetic changes as well as protein expression patterns in order to explore the roles of the Wnt pathway components, such as DKK1, DKK3, GSK3β, β-catenin, and APC in astrocytoma initiation and progression. Our findings on DKK1 and DKK3 show the importance of methylation in the regulation of Wnt signaling activity and also indicate pro-oncogenic effects of GSK3β on astrocytoma development and progression. Close connections between large deletions and mutations in the APC gene and increased β-catenin expression in glioblastoma were also established. Our results suggest that Wnt pathway related genes and proteins play an active role in the etiology of astrocytic brain tumors.
In the present study, we investigated genetic and epigenetic changes and protein expression levels of negative regulators of Wnt signaling, DKK1, DKK3, and APC as well as glycogen synthase kinase 3 (GSK3β) and β-catenin in 64 human astrocytomas of grades II–IV. Methylation-specific PCR revealed promoter methylation of DKK1, DKK3, and GSK3β in 38%, 43%, and 18% of samples, respectively. Grade IV comprised the lowest number of methylated GSK3β cases and highest of DKK3. 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 also analyzed. Spearman’s correlation confirmed the prevalence of β-catenin’s active form (rs = 0.634, p < 0.001) in astrocytoma tumor cells. The Wilcoxon test revealed that astrocytoma with higher levels of the active pGSK3β-Y216 form 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 the APC gene associated with increased β-catenin levels, together with oncogenic effects of both β-catenin and GSK3β, are clearly involved in astrocytoma evolution. Our findings contribute to a better understanding of the etiology of gliomas. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations. View Full-Text
Keywords: astrocytic brain tumors; Wnt signaling; DKKs; GSK3β; APC; β-catenin astrocytic brain tumors; Wnt signaling; DKKs; GSK3β; APC; β-catenin
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MDPI and ACS Style

Kafka, A.; Bukovac, A.; Brglez, E.; Jarmek, A.-M.; Poljak, K.; Brlek, P.; Žarković, K.; Njirić, N.; Pećina-Šlaus, N. Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma. Cancers 2021, 13, 2530. https://doi.org/10.3390/cancers13112530

AMA Style

Kafka A, Bukovac A, Brglez E, Jarmek A-M, Poljak K, Brlek P, Žarković K, Njirić N, Pećina-Šlaus N. Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma. Cancers. 2021; 13(11):2530. https://doi.org/10.3390/cancers13112530

Chicago/Turabian Style

Kafka, Anja, Anja Bukovac, Emilija Brglez, Ana-Marija Jarmek, Karolina Poljak, Petar Brlek, Kamelija Žarković, Niko Njirić, and Nives Pećina-Šlaus. 2021. "Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma" Cancers 13, no. 11: 2530. https://doi.org/10.3390/cancers13112530

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