Cancer Genomics & Proteomics. 2016 01-02;13(1): 31-46.
WNT/β-catenin signaling pathway and downstream modulators in low- and high-grade glioma
Tetyana Denysenko1,2, Laura Annovazzi1, Paola Cassoni3, Antonio Melcarne2, Marta Mellai1 and Davide Schiffer1
1Research Center / Policlinico di Monza Foundation, Vercelli, Italy;
2Department of Neurosurgery, CTO Hospital/Health and Science City, Turin, Italy;
3Department of Medical Sciences, University of Turin/Health and Science City, Turin, Italy
Background: Aberrant activation of the canonical Wingless-type MMTV integration site family (WNT)/β-catenin signaling pathway is critical for gliomas. Materials and Methods: In 74 gliomas of different histological grade and in 24 glioblastoma cell lines, protein expression of WNT member 3a (WNT3a), β-catenin and transcription factor 4 (TCF4) was investigated by immunohistochemistry, western blotting, immunofluorescence and immunocytochemistry. In tumors and cell lines, WNT3A expression was assessed at mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR). Results: WNT3a was overexpressed at protein and mRNA levels in malignant astrocytic tumors and cell lines. Cytoplasmic expression of β-catenin was detected in high-grade gliomas and cell lines, with evidence of nuclear translocation on fractionated protein extracts. Activating mutations in the β-catenin encoding gene (CTNNB1) were excluded by direct sequencing. TCF4 was statistically correlated with Ki-67/MIB-1 and cyclin D1 labeling indices. Conclusion: Expression of WNT3a, cytoplasmic β-catenin and TCF4 was significantly associated with the histological malignancy grade and with a worse prognosis for glioma patients.
The Wingless-type MMTV integration site family (WNT) signaling pathway is involved in different biological processes (embryonic development, cell polarity, fate and tissue homeostasis) (1). In particular, the β-catenin (CTNNB1)-dependent pathway (referred as “canonical”) plays a role in the development and progression of several human malignancies (2), including gliomas (3), through the key effectors β-catenin and Transcription Factor 4 (TCF4).
The signaling transduction cascade is triggered by the binding of WNT ligands to Frizzled (Fz) protein and to low density lipoprotein receptor-related proteins 5/6 (LRP5/6). It follows the cytoplasmic β-catenin accumulation and the subsequent nuclear translocation. In the nucleus, β-catenin binds to members of the TCF/lymphoid-enhancer-binding factor (LEF) family of transcription factors and modulates expression of target genes critical for cell proliferation, differentiation, survival and apoptosis, such as cyclin D1, FRA1, c-MYC, c-JUN and survivin. In the absence of WNT ligands, β-catenin is sequestrated into a degradation complex composed of axin, tumor suppressor adenomatous polyposis coli (APC), casein kinase Iα (CKIα) and glycogen synthase kinase-3β (GSK-3β) proteins. Upon phosphorylation by CKIα and GSK-3β, β-catenin is polyubiquitinated for the proteasomal degradation (Figure 1).
Figure 1. Scheme of the canonical Wnt/β-catenin signaling.
In the absence of WNT ligands, however, the oncogenic activation of β-catenin may result from genetic mutations affecting key components of the canonical pathway (APC, β-catenin encoding gene [CTNNB1], axin) (2).
The WNT/β-catenin signaling pathway is also involved in the maintenance of glioma stem cells by inhibiting differentiation, conditioning radio- (4) and chemoresistance (5), and inducing an invasive phenotype.
In this study we investigate the aberrant activation of the canonical pathway in a series of 74 low- and high-grade gliomas and in 24 glioblastoma (GBM) cell lines by determining the protein expression levels of the key players WNT3a, β-catenin and TCF4. They have been evaluated on formalin fixed and paraffin embedded (FFPE) tumor samples by immunohistochemistry, on cell lines by immunofluorescence and immunocytochemistry, on fresh frozen specimens and cell lines by Western blotting (WB) analysis. WNT3A expression was assessed at mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR) in both tumor samples and cell lines.
Twenty-four GBM cell lines were obtained from surgically resected GBMs as neurospheres (NS) or adherent cells (AC), accordingly to different culture conditions. Neurospheres, isolated in serum-free culture medium supplemented with growth factors, exhibit stemness properties and in vivo tumorigenicity; AC, isolated in serum, correspond to a more differentiated tumor cell status.
In this study, we focused on the WNT3a ligand since, unlike the other WNT ligands present in neuronal stem cells, it enhances proliferation and differentiation of neuronal progenitor cells in adult mice brain and is required for the development of the mammalian hippocampus. Moreover, previous studies reported WNT3a overexpression in high-grade gliomas and GBM stem cells (GSCs).
The immunohistochemical analysis of WNT3a revealed a slight expression on the cell membranes of anaplastic gliomas and a high expression in the cytoplasm and the cell membranes of GBMs (Figure 2). As a matter of fact, WNT3A up-regulation at mRNA level was demonstrated by qRT-PCR in anaplastic astrocytomas (2/2, 100%) and GBMs (7/11, 63.6%) with increasing fold change from grade III to grade IV (Figure 3). A weak and variable WNT3A expression was detected in low grade gliomas as well. The up-regulation rate observed in GBM tumor specimens was in agreement with previous data from The Cancer Genome Atlas, documenting WNT3A overexpression in 55% of glioma samples. In GBM cell lines, WNT3A up-regulation was detected in 8/24 cases (33.3%), with a similar distribution in NS (3/10, 30%) and AC (5/14, 35.7%) (Figure 3).
Figure 2. Activation of the WNT/β-catenin signaling pathway in glioma. Immunohistochemistry. A: GBM, diffuse cytoplasmic and membranous WNT3a expression, ×400. B: Id, high cytoplasmic and membranous β-catenin expression, ×400. C: Id, cytoplasmic and membranous β-catenin expression in proliferative endothelial cells, ×630. D: Id, high nuclear TCF4 expression in tumor cells, ×400. E: Id, Ki-67/MIB-1 positive nuclei in the same area as in (D), ×200. F: Id, cyclin D1 positive nuclei in the same area as in (D), ×200. All DAB. Immunofluorescence of GBM (NS) cell lines. G: High cytoplasmic WNT3a expression. H: High cytoplasmic β-catenin expression. I: High nuclear TCF4 expression. Nuclei were counterstained with DAPI. All ×200 magnification.
The crucial point in the pathway is the nuclear translocation of β-catenin. In FFPE tumor specimens, β-catenin was expressed in the cytoplasm and on the cell membranes more intensely with increasing histologic grade (Figure 2). The immunostaining intensity was higher and mainly cytoplasmic in high- than low-grade astrocytomas whereas it was limited to the cell membranes in oligodendroglial tumors. Despite of the lack of nuclear immunoreactivity for (non-phospho Ser33/37/Thr41) active β-catenin, rare, scattered and barely identifiable positive nuclei have been observed in both tumor and proliferative endothelial cells (Figure 2). As the cytoplasmic expression of β-catenin precedes the nuclear translocation and the subsequent transcription of downstream genes, we considered it indicative of pathway activation. However, WB analysis revealed β-catenin expression in the nuclear fractions obtained from fresh GBM tumor specimens and cell lines (Figure 4). In the normal nervous tissues (NNT), the expression of β-catenin was limited to the cell membranes, revealing more a structural than an oncogenic function.
Phospho-β-catenin (Ser33/37/Thr41), the destabilized and inactive form of β-catenin, showed a variable expression in all gliomas, that was inversely correlated to the histologic grade. Phospho-GSK-3β (Tyr216), the active form of GSK-3β in the degradation complex of β-catenin, exhibited a similar expression pattern, opposite to the that of WNT3a and β-catenin.
Among the critical target genes of the pathway, we studied the expression of the main effector TCF4. We found TCF4 expression in nuclei of tumor cells, mainly in high grade astrocytomas, and in nuclei of proliferative endothelial cells from microvascular proliferations. The immunostaining intensity was variable; at its maximum value, it was significantly associated to the histologic malignancy grade and to Ki-67/MIB-1 and Cyclin D1 labeling indices (LIs) (Figure 2). This finding suggests TCF4 as a potential proliferation marker in gliomas.
WNT3a, cytoplasmic β-catenin and TCF4 expression was detected neither in the NNT nor in pilocytic astrocytoma.
Immunofluorescence revealed in NS a variable expression of WNT3a in the cytoplasm, a more consistent expression of β-catenin in the cytoplasm and on the cell membranes and a constant expression of TCF4 in nuclei. In contrast, AC showed a slight expression of WNT3a and absence of β-catenin and TCF4 expression (Figure 2). The expression of WNT3a and β-catenin in cell lines was confirmed by WB analysis (Figure 4). Immunocytochemistry confirmed the expression patterns of the three key molecules and, in addition, revealed a nuclear staining for β-catenin in NS.
Notably, after exposure to 50 μM Temozolomide (TMZ) for 48 hours, WNT3a, β-catenin and TCF4 expression was revealed in AC. This finding demonstrates that TMZ induces aberrant pathway activation in AC, suggesting that its inhibition may enhance the response of GSCs. The expression of WNT3a, β-catenin and TCF4 did not change in NS after treatment.
Finally, we focused on the search for potential driver mutations in the β-catenin CTNNB1 gene by Sanger sequencing mutation analysis. The CTNNB1 exon 3 encodes the N-terminal regulatory sequence containing the critical phosphorylation sites for GSK-3β. The absence of CTNNB1 activating mutations excluded genetic events as responsible for the oncogenic activation of β-catenin in malignant astrocytic gliomas.
Figure 3. WNT3A up-regulation in gliomas and GBM cell lines by qRT-PCR. A: Relative WNT3A expression levels in glioma tumor samples of different type and histologic malignancy grade. B: Relative WNT3A expression levels in a panel of 24 GBM cell lines, cultured as NS or AC.
It can be hypothesized that the canonical WNT3a/β-catenin signaling contributes to tumorigenesis and progression of malignant gliomas and correlates with cell proliferation.
WNT3a, β-catenin and TCF4 expression levels were significantly correlated to the histologic malignancy grade. In patients affected by malignant astrocytoma, WNT3a and cytoplasmic/nuclear β-catenin immunoreactivity was associated to a worse prognosis.
Relevance of the study: This study showed the activation of Wnt/β-catenin signaling pathway in malignant astrocytic gliomas. The activation is mediated by the presence of WNT3a ligand that results significantly up-regulated at mRNA levels and overexpressed at protein level in all anaplastic astrocytomas and in a fraction of GBMs (63.6%). Cytoplasmic/nuclear translocation of β-catenin and expression of TCF4 occur in the same tumors as well. TCF4 significantly correlates with the proliferation markers Ki-67/MIB-1 and Cyclin D1.
Our findings may have important implications in the new immunotherapeutic strategies. A key issue in the clinical response to treatment is the possibility to develop a baseline T-cell response to the tumor that is indicated by the presence of infiltrating CD8+ T-cells in the tumor microenvironment. Aberrant activation of the WNT/β-catenin signaling can undermine the T-cell response to the tumor and thus mediate tumor immune evasion and resistance to immunotherapies, as for other solid tumors. In this regard, key effectors of the WNT/β-catenin pathway may represent new candidate targets to enhance the immune response to the tumor in patients with malignant glioma.
Figure 4. Western blotting analysis. A: Representative images of two independent experiments displaying WNT3a and β-catenin expression in whole cell extracts from GBM cell lines, cultured as NS (A) or AC (B). C: β-catenin expression in cytosolic and nuclear extracts from GBM cell lines and from tumor tissues (D).
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Marta Mellai, PhD
Research Center, Policlinico di Monza Foundation
Via Pietro Micca 29, 13100 Vercelli, Italy