Original ArticlesBlocking of the interaction between Wnt proteins and their co-receptors contributes to the anti-tumor effects of adenovirus-mediated DKK3 in glioblastoma
Introduction
Glioblastoma multiforme (GBM), the most common and lethal type of primary human brain tumor in adults, accounts for approximately 12%–15% of all intracranial tumors and for 50% of all gliomas. Despite the use of aggressive and multimodal therapies involving tumor resection, chemotherapy, adjuvant therapy, immunotherapy, and radiation therapy, GBM remains incurable [1], [2], [3]. Therefore, new, more effective adjuvant therapies with low toxicity and gene therapy are needed.
Various pathways are involved in glioma tumorigenesis, and the precise role of Wnt signaling remains to be elucidated. The Wnt signaling pathways are classically categorized as canonical and non-canonical (β-catenin-dependent and independent, respectively), and their combination is required to maintain the proliferative capacity of human GBM [4]. In the canonical pathway, Wnt3a, a member of the Wnt ligands, binds to frizzled receptors (Fzd) and the co-receptor low-density lipoprotein receptor-related receptor 6 (LRP6). This results in the stabilization of cytosolic β-catenin and facilitates its translocation into the nucleus to activate transcription factors [5]. In the non-canonical pathway, Wnt5a, another Wnt member, binds to Fzd and to the tyrosine kinase-like orphan receptor 2 (ROR2) and primarily modulates cell movements such as migration and invasion [6], [7]. In human malignant glioma, the gene and protein expressions of Wnt5a were higher than in normal human brain tissues and related to the malignancy grade [8]. However, the role and manner of the interaction of Wnt proteins with the receptors remain unclear.
Dickkopf (DKK) family members exhibit distinct overlapping expression patterns in mouse and human tissues, and their effects on Wnt signaling may be different [9]. DKK-1, -2, and -4 share the ability to inhibit the canonical (Wnt/β-catenin) pathway through binding to the co-receptor LRP5/6 with high affinity [10], but they are ineffective with respect to the non-canonical (Wnt/PCP and Wnt/Ca2+) pathway. The role of DKK1 through the Wnt/β-catenin pathway was reported in glioma [11]. The co-binding of DKKs to the co-receptor class of Kremen proteins (Krm-1/2) potentiates their ability to block Wnt signaling because this ternary complex leads to the internalization and degradation of LRP [2], [9], [10], [12] and they negatively regulate the expression of various Wnt target genes.
On the other hand, the third member of the DKK family (DKK3), which matches the reduced expression in the immortalized cell (REIC) gene (NCBI Gene ID 27122) and exhibits unique properties, does not bind to LRP5/6 [13]. In human prostate cancer cells, the adenovirus-mediated over-expression of DKK3 (Ad-DKK3) selectively induces apoptosis through the activation of c-Jun-NH2-kinase, suggesting the inhibition of the non-canonical Wnt pathway [14]. However, our group documented that DKK3 was down-regulated in GBM cells and that its over-expression from a plasmid vector drastically inhibited their growth via caspase-dependent apoptosis and the degradation of β-catenin [15]. This suggests inhibition of the canonical Wnt signal pathway by DKK3. Thus the role of DKK3 in Wnt signaling pathways may differ among tumor cell types. We used Ad-DKK3 to examine the anti-tumor effects of the DKK3 protein and its regulation of the Wnt signaling pathway.
Here we first demonstrate that both canonical and non-canonical Wnt signaling pathways are augmented in GBM cells and that the Wnt proteins Wnt3a and Wnt5a interact with both co-receptors LRP6 and ROR2. We also demonstrate that the DKK3 protein elicited by Ad-DKK3 treatment attenuates the expression of Wnt3a, Wnt5a, and LRP6 but not of ROR2 and inhibits the interaction of Wnt proteins with the receptors, thereby affecting both the canonical and non-canonical Wnt downstream cascades. We suggest that these effects play a role in the anti-tumor effects of Ad-DKK3.
Section snippets
Cell lines and human GBM
The human GBM cell lines U87MG and U251MG, and normal human astrocytes (NHA) were purchased from American Type Culture Collection (Manassas, VA, USA), the Health Science Research Resources Bank (Osaka, Japan) and Lonza Japan Co. (Tokyo, Japan), respectively. The TGB-111 cells were primary GBM cells from a patient who granted prior informed consent for their use in this study. All GBM cell lines were cultured in RPMI-1640 medium (Invitrogen, NJ, USA) with 10% fetal bovine serum (GIBCO-BRL, NY,
Ad-DKK3 inhibits the proliferation of GBM cells and induces intrinsic apoptosis
We first examined the effects of Ad-DKK3 and Ad-LacZ in GBM cells. Each GBM cell line was exposed to Ad-DKK3 or Ad-LacZ at a multiplicity of infection (MOI) of 10, 30, 100, and 300. At 120 hr post-treatment, Ad-DKK3, but not Ad-LacZ, decreased the viability of U87MG-, U251MG-, and TGB-111 cells in a dose-dependent manner; the 50% inhibition concentration of cell viability was around 100 MOI. For Ad-DKK3 at 100 MOI, the growth of all GBM cell lines was inhibited in a time-dependent manner (
Discussion
Our previous study showed that the level of both intracellular and extracellular DKK3 was low in GBM [15]. In contrast, Zhou et al. [16] reported that the high level of DKK1 protein in the cerebral fluid of glioma patients is associated with tumor malignancy. DKK1 functions as a tumor suppressor and as a pro-apoptotic factor in glioma [11] through the canonical- but not the non-canonical Wnt pathway. On the other hand, the antitumor effects and the role of DKK3 in Wnt pathways in GBM remained
Funding
This work was supported in part by a Grant-in-Aid for Scientific Research (No. 23592128) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Conflict of interest
No potential conflicts of interest were disclosed.
Acknowledgement
We are extremely grateful to Professor Hiromi Kumon and Dr. Masami Watanabe for the kind gift of Ad-LacZ and Ad-DKK3 and thoughtful advices. We thank Emiko Nishikawa and Aya Satoh for their excellent technical support.
References (32)
- et al.
Wnt/beta-catenin signaling: components, mechanisms, and diseases
Dev. Cell
(2009) - et al.
Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and frizzled homologs and promotes ventral cell fates in Xenopus
J. Biol. Chem
(2000) - et al.
Cell/tissue-tropic functions of Wnt5a signaling in normal and cancer cells
Trends Cell Biol
(2010) - et al.
Role of Wnt5a in the proliferation of human glioblastoma cells
Cancer Lett
(2007) - et al.
Functional and structural diversity of the human Dickkopf gene family
Gene
(1999) - et al.
PRDM1 is directly targeted by miR-30a-5p and modulates the Wnt/β-catenin pathway in a Dkk1-dependent manner during glioma growth
Cancer Lett
(2013) - et al.
Silibinin inhibits Wnt/beta-catenin signaling by suppressing Wnt co-receptor LRP6 expression in human prostate and breast cancer cells
Cell. Signal
(2012) - et al.
FoxM1 promotes beta-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis
Cancer Cell
(2011) - et al.
Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6
J. Biol. Chem
(2010) - et al.
Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling
Cell
(2008)
Chemoradiotherapy in malignant glioma: standard of care and future directions
J. Clin. Oncol
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma
N. Engl. J. Med
The art of gene therapy for glioma: a review of the challenging road to the bedside
J. Neurol. Neurosurg. Psychiatry
Dishevelled 2 signaling promotes self-renewal and tumorigenicity in human gliomas
Cancer Res
Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins
Mol. Cell. Biol
LDL-receptor-related protein 6 is a receptor for Dickkopf proteins
Nature
Cited by (19)
Non-coding RNAs (ncRNAs) and multidrug resistance in glioblastoma: Therapeutic challenges and opportunities
2024, Pathology Research and PracticeWNT5B in cellular signaling pathways
2022, Seminars in Cell and Developmental BiologyMechanisms regulating glioma invasion
2015, Cancer LettersCitation Excerpt :In the calcium pathway, various Wnt and Fz homologs activate calcium/calmodulin-dependent kinase II and protein kinase C [33]. These pathways have been shown to be upregulated in GBMs and are known to be activated by Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, and Wnt11 [31,34]. The aberrant activation of the Wnt pathway promotes cancer progression in many cancer types [30].
The Wnt/β-catenin signalling pathway in Haematological Neoplasms
2022, Biomarker ResearchHigh DKK3 expression related to immunosuppression was associated with poor prognosis in glioblastoma: machine learning approach
2022, Cancer Immunology, ImmunotherapyHypoxia-Induced miR-137 Inhibition Increased Glioblastoma Multiforme Growth and Chemoresistance Through LRP6
2021, Frontiers in Oncology