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Wnt/Β-Catenin Signaling Pathway Induces Autophagy

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Wnt/Β-Catenin Signaling Pathway Induces Autophagy Yun et al. Cell Death and Disease (2020) 11:771 https://doi.org/10.1038/s41419-020-02988-8 Cell Death & Disease ARTICLE Open Access Wnt/β-catenin signaling pathway induces autophagy-mediated temozolomide-resistance in human glioblastoma Eun-Jin Yun 1,SangwooKim2,Jer-TsongHsieh3,4 and Seung Tae Baek 5,6 Abstract Temozolomide (TMZ) is widely used for treating glioblastoma multiforme (GBM), however, the treatment of such brain tumors remains a challenge due to the development of resistance. Increasing studies have found that TMZ treatment could induce autophagy that may link to therapeutic resistance in GBM, but, the precise mechanisms are not fully understood. Understanding the molecular mechanisms underlying the response of GBM to chemotherapy is paramount for developing improved cancer therapeutics. In this study, we demonstrated that the loss of DOC-2/DAB2 interacting protein (DAB2IP) is responsible for TMZ-resistance in GBM through ATG9B. DAB2IP sensitized GBM to TMZ and suppressed TMZ-induced autophagy by negatively regulating ATG9B expression. A higher level of ATG9B expression was associated with GBM compared to low-grade glioma. The knockdown of ATG9B expression in GBM cells suppressed TMZ-induced autophagy as well as TMZ-resistance. Furthermore, we showed that DAB2IP negatively regulated ATG9B expression by blocking the Wnt/β-catenin pathway. To enhance the benefit of TMZ and avoid therapeutic resistance, effective combination strategies were tested using a small molecule inhibitor blocking the Wnt/ β-catenin pathway in addition to TMZ. The combination treatment synergistically enhanced the efficacy of TMZ in GBM cells. In conclusion, the present study identified the mechanisms of TMZ-resistance of GBM mediated by DAB2IP 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; and ATG9B which provides insight into a potential strategy to overcome TMZ chemo-resistance. Introduction different positions, which can interfere with DNA repli- Glioblastoma multiforme (GBM) is the most prevalent cation and induce cell cycle arrest at G2/M, consequently primary malignant brain tumor and, remains one of the leading to apoptosis3,4. Concomitant therapy using TMZ most lethal cancers, with a median survival of only and radiation has improved the prognosis for GBM 15 months upon diagnosis1. The standard care for GBM patients. However, the average survival expectancy includes maximally safe surgical resection followed by remains 14.6 months, including 2.5 months of increased radiation therapy and concurrent temozolomide (TMZ) median survival2,5. GBMs eventually relapse after treat- chemotherapy2. TMZ is a DNA alkylating agent known to ments, moreover, not all patients respond to this therapy induce several DNA adducts by methylating DNA at mainly due to TMZ-resistance, and the chemo-resistance remains one of the major clinical challenges2,5. Therefore, understanding the molecular mechanisms contributing to Correspondence: Eun-Jin Yun ([email protected]) or Seung this resistant phenotype will provide important insight for Tae Baek ([email protected]) developing novel therapeutic approaches and identifying 1POSTECH Biotech Center, POSTECH, Pohang, Republic of Korea 2Department of Biomedical Systems Informatics and Brain Korea 21 PLUS those patients most likely to benefit from a specific Project for Medical Science, Yonsei University College of Medicine, Seoul, treatment regimen. The overexpression of the demethy- Republic of Korea lating enzyme O6-methylguanine-DNA methyltransferase Full list of author information is available at the end of the article 6 These authors contributed equally: Eun-Jin Yun, Seung Tae Baek (MGMT), removing alkyl groups from the O position of Edited by B. Zhivotovsky © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Yun et al. Cell Death and Disease (2020) 11:771 Page 2 of 10 Fig. 1 TMZ-resistant GBM cell lines express a low level of DAB2IP. a TMZ log–dose–response analysis (IC50) in four human glioblastoma cell lines. Cells were treated with increment doses of TMZ as indicated for 48h, and IC50 was determined with cytotoxicity results assessed by MTT assay. Percent survival curves after TMZ treatment was constructed by plotting the cell survival whose values indicate mean ± SD (n = 5). Error bars indicate standard deviation. b The mRNA expression of DAB2IP analyzed by real-time PCR in four human glioblastoma cell lines. Black and white bars indicate DAB2IP-high and DAB2IP-low cell lines, respectively. Means ± SD; n = 3; Student’s two-tailed t-test, **p < 0.01. c Representative images of western blot against DAB2IP and β-actin. guanine directly, has been implicated as one mechanism autophagy by negatively regulating ATG9B via the Wnt/ of TMZ-resistance6. While MGMT remains a major cause β-catenin pathway. Combination treatment of GBM cells of TMZ-resistance, some MGMT-deficient GBMs are still using a small molecule inhibitor blocking the Wnt/ resistant to TMZ suggesting that other components may β-catenin pathway in addition to TMZ synergistically also be involved in TMZ-resistance. However, the enhanced the efficacy of TMZ. Our results showed that underlying molecular mechanisms remain unclear3,4,6. DAB2IP could function as a chemo-sensitizer to TMZ in Meanwhile, the DOC-2/DAB2 interacting protein combination treatment by suppressing Wnt/β-catenin- DAB2IP was previously identified as a member of the Ras- mediated autophagy. GTPase activating protein family, and functions as a Overall, these findings demonstrate a link between the tumor suppressor in cancer progression7,8. In various loss of DAB2IP and TMZ-resistance mechanisms and cancer types, DAB2IP has been shown to control cell offer a potential therapeutic strategy to re-sensitize proliferation, apoptosis, and cell survival by inhibiting the chemo-resistant GBM. Ras/MAPK pathway, activating the Ask1/JNK pathway, and inhibiting the PI3K/Akt pathway, respectively7,9,10.As Results a Ras-GAP, DAB2IP is highly expressed in developing and DAB2IP is downregulated in TMZ-resistant glioblastoma adult brains, where it has been shown to interact with cells Reelin transducing protein Dab2, and function in reg- The IC50 values for TMZ in GBM cell lines vary in many ulating synaptogenesis, dendritic morphogenesis, and studies depending on the experimental conditions4. Thus – neuronal differentiation and migration8,11 13. However, we determined the responsiveness of GBM cells to TMZ the physiological and pathological function of DAB2IP in in our system. As shown in Fig. 1a, the IC50 for TMZ GBM is relatively unknown. Previously, we demonstrated ranged from 61.8 ± 7.9 to 225.1 ± 17.5 μM and fell into that the loss of DAB2IP expression resulted in increased two groups, the TMZ-sensitive (A172 and U373; chemo-resistance as well as radioresistance in some can- <100 μM) and the TMZ-resistant groups (LN18 and – cer types14 16. Interestingly, we observed that the loss of LN229; >200 μM). We then compared the expression DAB2IP in GBM cells increased their chemo-resistance pattern of DAB2IP in the TMZ-sensitive and TMZ- to TMZ. resistant cells. DAB2IP, a novel tumor suppressor, is often Here, we demonstrated a link between the loss of downregulated in advanced stages of many types of can- DAB2IP and TMZ-resistance. Mechanistically, DAB2IP cer, and the loss of DAB2IP expression has been shown to sensitized GBM to TMZ and suppressed TMZ-induced correlate with the poor prognosis and increased tumor Official journal of the Cell Death Differentiation Association Yun et al. Cell Death and Disease (2020) 11:771 Page 3 of 10 metastasis of many malignancies7,8. In addition, according phase arrest after TMZ treatment, whereas the knock- to previous studies, DAB2IP has critical roles in sup- down of DAB2IP decreased G2/M arrest (Supplementary pressing chemo-resistance and radioresistance in different Fig. S1). – types of cancer14 16. To investigate whether DAB2IP is In addition, the depletion of DAB2IP significantly pro- involved in TMZ sensitivity in GBM as well, we screened moted the inhibitory effect of TMZ on cell proliferation the expression level of DAB2IP in GBM cell lines that and colony formation (Fig. 2c). Since DAB2IP expression respond differently to TMZ. We observed TMZ-resistant levels were not significantly affected by TMZ treatment cells expressed a low level of DAB2IP in both RNA and (Supplementary Fig. S2), the manipulation of DAB2IP protein levels (Fig. 1b, c) indicating a possible link levels seemed to regulate TMZ sensitivity directly. between the level of DAB2IP and chemo-resistance to Together, these results indicate that DAB2IP expression TMZ in GBM cell lines. may be inversely correlated with
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