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01-12-2020 | Glioblastoma | Research article

Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells

Authors: Fengyu Zhang, Ruilai Liu, Haishi Zhang, Cheng Liu, Chunfang Liu, Yuan Lu

Published in: BMC Cancer | Issue 1/2020

Abstract

Background

Glioblastoma is devastating cancer with a high frequency of occurrence and poor survival rate and it is urgent to discover novel glioblastoma-specific antigens for the therapy. Cancer-germline genes are known to be related to the formation and progression of several cancer types by promoting tumor transformation. Dazl is one such germline gene and is up-regulated in a few germ cell cancers. In this study, we analyzed the expression of Dazl in human glioblastoma tissues and cells, and investigated its significance in proliferation, migration, invasion and chemoresistance of the glioblastoma cell lines.

Methods

We evaluated the expression of Dazl in different pathologic grades of glioblastoma tissues by immunohistochemistry. We assessed the expression of Dazl in glioblastoma cells and normal human astrocytes (NHA) cells by western blotting and RT-qPCR. Then we generated Dazl knockout glioblastoma cell lines using the CRISPR/Cas9 gene-editing technology to explore the cellular function of Dazl. We detected the proliferation and germline traits via CCK-8 assays and alkaline phosphatase staining, respectively. Boyden chamber assays were performed to measure glioblastoma cell migration and invasion. Crystal violet staining was used to determine the number of viable cells after the treatment of Doxorubicin and Temozolomide. Finally, we used subcutaneous xenograft studies to measure the growth of tumors in vivo.

Results

We found that Dazl was upregulated in glioblastoma tissues and glioblastoma cell lines. Dazl knockdown glioblastoma cells showed decreased cellular proliferation, migration, invasion, and resistance in vitro, and inhibited the initiation of glioblastoma in vivo. The glioblastoma cell lines A172, U251, and LN229 were found to express stem cell markers CD133, Oct4, Nanog, and Sox2. The expression of these markers was downregulated in Dazl-deficient cells.

Conclusions

Our results indicated that Dazl contributes to the tumorigenicity of glioblastoma via reducing cell stemness. Therefore, cancer-germline genes might represent a new paradigm of glioblastoma-initiating cells in the treatment of malignant tumors.

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Huang W, Zhong Z, Luo C, Xiao Y, Li L, Zhang X, Yang L, Xiao K, Ning Y, Chen L, et al. The miR-26a/AP-2alpha/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma. Theranostics. 2019;9(19):5497–516.PubMedPubMedCentral

Xi G, Best B, Mania-Farnell B, James CD, Tomita T. Therapeutic potential for bone morphogenetic protein 4 in human malignant Glioma. Neoplasia. 2017;19(4):261–70.PubMedPubMedCentral

Wang B, Wang M, Zhang W, Xiao T, Chen C-H, Wu A, Wu F, Traugh N, Wang X, Li Z, et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat Protoc. 2019;14(3):756–80.PubMedPubMedCentral

López-Valero I, Saiz-Ladera C, Torres S, Hernández-Tiedra S, García-Taboada E, Rodríguez-Fornés F, Barba M, Dávila D, Salvador-Tormo N, Guzmán M, et al. Targeting Glioma initiating cells with a combined therapy of cannabinoids and temozolomide. Biochem Pharmacol. 2018;157:266–74.PubMed

Kuo Y-C, Chang Y-H, Rajesh R. Targeted delivery of etoposide, carmustine and doxorubicin to human glioblastoma cells using methoxy poly (ethylene glycol)-poly(ε-caprolactone) nanoparticles conjugated with wheat germ agglutinin and folic acid. Mater Sci Eng C Mater Biol Appl. 2019;96:114–28.PubMed

Chattergoon NN, D'Souza FM, Deng W, Chen H, Hyman AL, Kadowitz PJ, Jeter JR Jr. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2005;288(1):L202–11.

Auffinger B, Spencer D, Pytel P, Ahmed AU, Lesniak MS. The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence. Expert Rev Neurother. 2015;15(7):741–52.PubMedPubMedCentral

Lucena-Cacace A, Otero-Albiol D, Jiménez-García MP, Peinado-Serrano J. Carnero a.NAMPT overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis. Oncotarget. 2017;8(59):99514–30.PubMedPubMedCentral

Virolle T. Cancer stem cells in glioblastoma. Bull Cancer. 2017;104(12):1075–9.PubMed

10.

Hattermann K, Flüh C, Engel D, Mehdorn HM, Synowitz M, Mentlein R, Held-Feindt J. Stem cell markers in glioma progression and recurrence. Int J Oncol. 2016;49(5):1899–910.PubMed

11.

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.PubMed

12.

Li X, Hughes SC, Wevrick R. Evaluation of melanoma antigen (MAGE) gene expression in human cancers using the Cancer genome atlas. Cancer Genet. 2015;208(1):25–34.PubMed

13.

Van Tongelen A, Loriot A, De Smet C. Oncogenic roles of DNA hypomethylation through the activation of cancer-germline genes. Cancer Lett. 2017;396:130–7.PubMed

14.

Janic A, Mendizabal L, Llamazares S, Rossell D, Gonzalez C. Ectopic expression of germline genes drives malignant brain tumor growth in drosophila. Science. 2010;330(6012):1824–7.PubMed

15.

Chen H-H, Welling M, Bloch DB, Muñoz J, Mientjes E, Chen X, Tramp C, Wu J, Yabuuchi A, Chou Y-F, et al. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells. Stem Cell Reports. 2014;3(5):892–904.PubMedPubMedCentral

16.

Lin Y, Gill ME, Koubova J, Page DC. Germ cell-intrinsic and -extrinsic factors govern meiotic initiation in mouse embryos. Science. 2008;322(5908):1685–7.PubMed

17.

Gill ME, Hu Y-C, Lin Y, Page DC. Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells. Proc Natl Acad Sci U S A. 2011;108(18):7443–8.PubMedPubMedCentral

18.

Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, Dorin J, Cooke HJ. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature. 1997;389(6646):73–7.PubMed

19.

Schrans-Stassen BH, Saunders PT, Cooke HJ, de Rooij DG. Nature of the spermatogenic arrest in Dazl −/− mice. Biol Reprod. 2001;65(3):771–6.PubMed

20.

Zhang J, Tam W-L, Tong GQ, Wu Q, Chan H-Y, Soh B-S, Lou Y, Yang J, Ma Y, Chai L, et al. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol. 2006;8(10):1114–23.PubMed

21.

Luo G, Luo W, Sun X, Lin J, Wang M, Zhang Y, Luo W, Zhang Y. MicroRNA-21 promotes migration and invasion of glioma cells via activation of Sox2 and β-catenin signaling. Mol Med Rep. 2017;15(1):187–93.PubMed

22.

Abdelrahman AE, Ibrahim HM, Elsebai EA, Ismail EI, Elmesallamy W. The clinicopathological significance of CD133 and Sox2 in astrocytic glioma. Cancer Biomark. 2018;23(3):391–403.PubMed

23.

Guo Y, Liu S, Wang P, Zhao S, Wang F, Bing L, Zhang Y, Ling EA, Gao J, Hao A. Expression profile of embryonic stem cell-associated genes Oct4, Sox2 and Nanog in human gliomas. Histopathology. 2011;59(4):763–75.PubMed

24.

Sedaghat S, Gheytanchi E, Asgari M, Roudi R, Keymoosi H, Madjd Z. Expression of Cancer stem cell markers OCT4 and CD133 in transitional cell carcinomas. Appl Immunohistochem Mol Morphol. 2017;25(3):196–202.PubMed

25.

Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000;24(4):372–6.PubMed

26.

Du Y. The role and molecular mechanism of DAZL gene in lung cancer. Hunan Normal University. 2019;16–36. [in Chinese].

27.

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308.PubMedPubMedCentral

28.

Li XT, Zhou YX, Du ZW. Novel lncRNA-ZNF281 regulates cell growth, stemness and invasion of glioma stem-like U251s cells. Neoplasma. 2019;66(1):118–27.PubMed

29.

Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell. 2006;124(6):0–1115.

30.

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819.PubMedPubMedCentral

31.

Wang B, Wang M, Zhang W, Xiao T, Chen C-H, Wu A, et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat Protoc. 2019;14(3):756–80.PubMedPubMedCentral

32.

Huang K, Yang C, Wang Q-X, Li Y-S, Fang C, Tan Y-L, et al. the CRISPR/Cas9 system targeting EGFR exon 17 abrogates NF-κB activation via epigenetic modulation of UBXN1 in EGFRwt/vIII glioma cells. Cancer Lett. 2017;388:269–80.PubMed

33.

Masui K, Cloughesy TF, Mischel PS. Review: molecular pathology in adult high-grade gliomas: from molecular diagnostics to target therapies. Neuropathol Appl Neurobiol. 2012;38(3):271–91.PubMedPubMedCentral

34.

Batash R, Asna N, Schaffer P, Francis N, Schaffer M. Glioblastoma Multiforme, diagnosis and treatment; recent literature review. Curr Med Chem. 2017;24(27):3002–9.PubMed

35.

Gzell C, Back M, Wheeler H, Bailey D, Foote M. Radiotherapy in Glioblastoma: the past, the present and the future. Clin Oncol (R Coll Radiol). 2017;29(1):15–25.

36.

Sahebjam S, Sharabi A, Lim M, Kesarwani P, Chinnaiyan P. Immunotherapy and radiation in glioblastoma. J Neuro-Oncol. 2017;134(3):531–9.

37.

Old LJ. Cancer vaccines: an overview. Cancer Immun. 2008;8(Suppl 1):1.PubMed

38.

Wang J, Rousseaux S, Khochbin S. Sustaining cancer through addictive ectopic gene activation. Curr Opin Oncol. 2014;26(1):73–7.PubMed

39.

Lee HC, Choi HJ, Lee HG, Lim JM, Ono T. Han JY.DAZL expression explains origin and central formation of primordial germ cells in chickens. Stem Cells Dev. 2016;25(1):68–79.PubMed

40.

Stefanidis K, Pergialiotis V, Christakis D, Patta J, Stefanidi D. Loutradis D.OCT-4 and DAZL expression in precancerous lesions of the human uterine cervix. J Obstet Gynaecol Res. 2015;41(5):763–7.PubMed

41.

Zagore LL, Sweet TJ, Hannigan MM, Weyn-Vanhentenryck SM, Jobava R, Hatzoglou M, Zhang C. Licatalosi DD.DAZL regulates germ cell survival through a network of PolyA-Proximal mRNA interactions. Cell Rep. 2018;25(5):1225–1240.e1226.PubMedPubMedCentral

42.

Li M, Zhu F, Li Z, Hong N, Hong Y. Dazl is a critical player for primordial germ cell formation in medaka. Sci Rep. 2016;6:28317.PubMedPubMedCentral

43.

Lin Y, Page DC. Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice. Dev Biol. 2005;288(2):309–16.PubMed

44.

Simpson AJG, Caballero OL, Jungbluth A, Chen Y-T, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer. 2005;5(8):615–25.PubMed

Title: Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells
Authors: Fengyu Zhang
Ruilai Liu
Haishi Zhang
Cheng Liu
Chunfang Liu
Yuan Lu
Publication date: 01-12-2020
Publisher: BioMed Central
Keywords: Glioblastoma
Glioblastoma
Glioblastoma
Published in: BMC Cancer / Issue 1/2020
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-020-07155-y

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