Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells

Glioblastoma is among the most prevalent primary brain tumor, accounting for 15–20% of all intracranial tumors. The median survival time is only 15 months. Among these, glioblastoma is characterized by excessive proliferation, high invasion and high resistance to clinical treatment [1‐3]. The current standard treatment for glioblastoma patients involves radical surgical resection followed by adjuvant radiation and chemotherapy, numerous antineoplastic drugs such as Doxorubicin (Dox) and Temozolomide (TMZ), are widely used as in clinical treatment of glioblastoma [4, 5]. However, glioblastoma is notorious for its chemoresistance to treatment, and despite many efforts have been made, the addition of Dox and TMZ against glioblastoma have largely failed. Recurrence after chemo- and radiotherapy is inevitable and eventually leads to high mortality in patients with glioblastoma [6]. Tumor initiation, therapeutic resistance, and recurrence originate from cancer-initiating cells (CICs) [7‐9]. CICs display some stem cell markers and exhibit sustained self-renewal. Glioblastoma cells with stem characteristics have been isolated from glioblastoma tissues or established glioblastoma cell lines, based on the expression of stem cell markers and the ability to survive in certain stem cell circumstances. Glioblastoma-initiating cells have been found to exhibit resistance to chemotherapy and radiotherapy, tumor-initiating potential, migration, and proliferative capacity [10].

Generally, the concepts of how CICs gain their ability to self-renew and proliferate are hardly understood. In the past decade, Takahashi [11] found that cancer cells could gain the embryonic characteristics enabling self-renew, which might be comparable to the reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs) by introducing embryonic stem cell transcription factors. Meanwhile, cancers acquire characteristic properties by reactivating genes normally expressed in embryonic and fetal life. The description of cancer-embryonic genes like CEA, the anomalous production of human chorionic gonadotrophin by a range of histologically distinct cancers, and the finding that germline genes are involved in the process of invasion and metastases [12, 13]. Previous work focusing on germline traits in cancers led to the discovery of cancer-germline (CG) genes, also called cancer-testis (CT) genes, which are mainly expressed in germline cells and are barely expressed in somatic adult tissues; however, they are abnormally activated in a wide variety of tumors [14]. Some of these human CG genes are suspected to be involved in the germline traits of oncogenesis, such as invasiveness, metastasis, immortality, angiogenesis, and hypomethylation, so they are being studied as biomarkers for cancers [14]. Dazl (Deleted in azoospermia-like), a member of the DAZ (Deleted in Azoospermia) gene family, which is also identified as a marker for germ cell identification [15]. Dazl is conserved in all vertebrates and acts as a meiosis-promoting factor in developing germ cells [16]. It is also a “licensing factor” that is required for primordial germ cells (PGCs) sexual differentiation [17]. Dazl can directly regulate apoptosis in PGCs by suppressing the translation of Caspase RNAs, loss of Dazl expression results in apoptosis of the postmigratory germ cells and infertility in both sexes in mice, with germ cell loss during development and a final block at meiosis [18, 19]. During the transition of PGCs into germ cells, Dazl acts as a translational regulator and regulates the transcription of the stemness genes Sox2, Sall4, and Suz12 [15, 20]. Sox2 regulates proliferation, migration, invasion, and colony formation of glioblastoma cells [21, 22]. CD133, Oct4, and Nanog are identified as stem/progenitor cell markers of glioblastoma [10] and participate in the tumorigenesis of astrocytic glioblastoma [22‐25]. Moreover, Dazl identified as a novel cancer germline gene and could promote the proliferation and resistance to chemical drugs of lung cancer cells by enhancing the translation of RRM2 [26]. However, whether Dazl is involved in the formation of glioblastoma has not been reported. Herein, to explore the correlation of Dazl expression and the tumorigenesis of glioblastoma, we generated glioblastoma Dazl^+/− GBM cell lines using the CRISPR/Cas9 gene editing system, and we evaluated that the Dazl knockdown attenuated cell proliferation, reduced cell migration, invasion, and chemo-resistance. These results support the concept that Dazl may be a cancer-germline gene involved in the development of human glioblastoma cells.

CRISPR/Cas9 technology is a powerful method for targeting desired genomic sites for gene editing or activity modulation via specific single-guide RNAs (sgRNAs) [30]. In the experiment, Dazl-sgRNAs were designed, synthesized and cloned into a lentiviral vector, which was subsequently transduced into glioma cells at a low multiplicity of infection to ensure that only one sgRNA copy was integrated per cell; then, the Cas9 enzyme was guided to the Dazl gene location, where Cas9 induced a double-strand break [31] The repair of such a break by glioma cells led to a knockout of the targeted Dazl gene, and the Dazl^+/− GBM cell lines grew stably for generations. CRISPR knockout technology has been highly effective in identifying genes that have functions in tumorigenesis. CRISPR/Cas9 is the most commonly applied method for generating clinical trials of human cancer [32], and it is far superior to the previously reported RNA interference technology because it ensures the functional stability of the Dazl gene in the cell inheritance.

Glioblastoma is one of the most malignant primary brain tumors associated with poor prognosis and low median survival [33, 34]. Glioblastoma is not a surgically curable disease because the glioblastoma cells invade the surrounding brain tissue and are among the most resistant to chemotherapy [35, 36]. Therefore, new targets in molecular knowledge, prognosis factor, and treatment are urgent. The similarity of the biological characteristics of cancer cells and germ cells prompted Lloyd J. Old to discover cancer/testis (CT) antigens [37]. The discovery elaborated a theory that aberrant expression of germline genes in cancers reflects the activation of the silenced gametogenic programme in somatic cells, and this programmatic acquisition is one of the driving forces of tumorigenesis. Extensive data have been assembled concerning the ectopic activation of germline genes in the progression of several human cancer types [38]. Dazl is responsible for germline traits and plays a central role in controlling pluripotency, differentiation, and apoptosis [15]. In this study, we demonstrated that ectopic expression of the germline gene Dazl in human glioblastoma and its association with tumorgenicity. We found that Dazl promotes cell proliferation in GBM since A172, U251, and LN229 GBM cells with Dazl knockdown exhibited a reduced cell proliferation rate (Fig. 2). We also showed that Dazl increases the ability of migration and invasion through the transwell assays (Fig. 3). Also, TMZ and Dox treated cell lines showed increased apoptosis in A172, U251, and LN229 GBM cells with Dazl knockdown (Fig. 4), which suggested the anti-apoptosis function of Dazl in GBM cells. Lastly, a screening of stem cell markers found that their expression decreased significantly in Dazl-knockdown cells (Fig. 5), suggesting the involvement of Dazl in the maintenance of the glioblastoma stem cell population.

Our work successfully discovered the relationship between Dazl and the proliferation of GBM cells. Dazl has been known for its involvement in cell proliferation in the integrity of PGCs in many vertebrates [39], Dazl is involved in the early proliferation of the germ cells [40], and also have essential roles in controlling a network of cell-cycle regulatory genes such as sox3 and Atm [41]. Dazl enhances postnatal germ cell survival via poly A-proximal interactions that promote the cell-cycle regulation and germ cell survival [41]. Dazl can also improve the spermatogonia proliferation via increasing steady-state levels of inherently unstable mRNA to ensure the high concentrations of regulatory factors in the germ cell development [42]. In this work, we found that Dazl was upregulated in GBM cells and glioblastoma tissues, especially in late-stage. These findings support the oncogenic function of Dazl in tumor formation and proliferation.

Besides regulating cell proliferation, Dazl was also found to be responsible for anti-apoptosis in GBM cells in this work. We found that multiple the number of GBM cells with Dazl knockdown experienced apoptosis compared to normal GBM cells under drug treatment (Fig. 4). Dazl regulates the expression of the key caspases, reveals a meaningful fail-safe mechanism that prevents stray PGCs from forming teratomas by sensitizing them to apoptotic cell death [15, 39, 43]. A previous study also demonstrated that the loss of PGCs in the Dazl^−/− embryo is due to increased apoptosis [43] and Dazl knockdown in PGCs causes increased apoptosis. Therefore, the silencing of Dazl induced drug susceptibility in glioblastoma cells by increasing apoptosis. Stemness is thought to be the main reason for chemoresistance, then we detected whether Dazl could regulate the stemness marker in glioblastoma.

Finally, we found that GBM cell lines A172, U251, and LN229 all expressed stemness markers CD133, Nanog, Oct4, and Sox2. Dazl-knocked down cell lines showed significantly decreased expression of these markers. Interestingly, at the transition of PGCs, Dazl-mediated silencing of both pluripotency factors and the polycomb complex allows PGCs to reduce the risk of teratoma formation by inhibition of the pluripotent program while simultaneously preventing somatic differentiation [15]. Dazl likely plays different roles in different developmental stages, and its role in a specific tissue remains the same in both normal and tumor cells. This findings confirm the theory that CG genes could exist in the GBM cells, and mainly present in reproductive tissues, such as testes, fetal ovaries, and trophoblasts, and are aberrantly expressed in a range of human cancers, but have limited expression on other normal tissues in adults [44]. Our results further demonstrated the cancer-promoting role of Dazl in glioblastoma cells and helped expand the knowledge that the germline gene could involve in the formation of glioblastomas. The tumor-suppressive effect of Dazl was exerted through inhibiting the transcriptional activity of Oct4, Sox2, and Nanog gene to attenuate the stemness and resistance of glioblastoma cells. However, our results do not discover the detailed mechanism of the Dazl regulates the tumorigenicity and stemness in glioblastoma cells. Further studies on Dazl expression and its function on these stemness markers should prove beneficial. Moreover, the relationship between germline genes and the tumorigenicity merits further investigation as they are involved in several important cellular signaling pathways. However, our study demonstrated that Dazl promotes the expression of stem cell markers and apoptosis of the GBM cells not through the WNT/beta-Catenin pathway.

In conclusion, Dazl functions as a novel cancer germline gene to initiate the stemness of glioblastoma cells by regulating the CD133/Oct4/Nanog/Sox2 regulatory axis and increasing the resistance of glioblastoma cells to Dox and TMZ. Additionally, Dazl knockdown not only promotes the glioblastoma cells proliferation, migration, and invasion in vitro, but also inhibits the initiation of glioblastoma in vivo. Therefore, understanding the underlying mechanisms of the cancer-germline gene in glioblastoma has new implications in future therapies to inhibit glioblastoma progression and recurrence.