Research ArticleExperimental Studies

The Role of ZEB2 Expression in Pediatric and Adult Glioblastomas

JAE KYUNG MYUNG, SEUNG AH CHOI, SEUNG-KI KIM, SEONG IK KIM, JIN WOO PARK and SUNG-HYE PARK
Anticancer Research January 2021, 41 (1) 175-185; DOI: https://doi.org/10.21873/anticanres.14763

Abstract

Background/Aim: Both pediatric glioblastoma (pGB) and adult glioblastoma (aGB) are clinically devastating, and are known to have different molecular pathogenesis. Here, we focused on the role of ZEB2 in pGB and aGB. Materials and Methods: Following transfection with ZEB2 siRNA into pGB cells (KNS42) and aGB cells (U87 and U373), cell proliferation, migration and invasion, and cell cycle progression were evaluated. Results: Targeted inhibition of ZEB2 induced up-regulation of E-cadherin expression and down-regulation of vimentin expression. Furthermore, it reduced invasion and migration of both pGB and aGB cells. Interestingly, in pGB cells, but not in aGB cells, silencing of ZEB2 reduced cell proliferation and viability, and affected the cell cycle progression of tumor cells. Conclusion: Inhibition of ZEB2 altered the mesenchymal features and reduced the migration and invasive ability of both pGB and aGB cells. ZEB2 effects were different in pGB and aGB cells regarding proliferation and cell cycle progression, suggesting that different underlying molecular mechanisms drive progression in these two types of tumors.

Key Words:

Glioblastomas (GB) are poorly differentiated astrocytic tumors, which are the most common high grade glioma in adults, but rare in children (1).

Although recent advances in cancer treatment have extended survival for many cancer patients, the prognosis for GBs remains poor. Pediatric GB (pGB) and adult GB (aGB) share morphological characteristics. However, the molecular pathologies of these two entities are different (2). Unlike aGB, EGFR gene amplification and EGFRvIII mutations and deletions are rare in pGB (1-3). Besides, the rate of PDGFR-α gene amplification is lower in pGB compared to aGB (4). Although different molecular pathologies may characterize these two types of GBs, their clinical outcome is similar because they are eventually resistant to chemotherapy, radiation therapy, or both (1). Epithelial-to-mesenchymal transition (EMT) is a complex mechanism contributing to the aggressive progression of diverse epithelial tumors as well as to certain physiological mechanisms in organ development (5, 6). This process enhances the acquisition of an invasive phenotype and stem cell characteristics by cancer cells (7). Also, EMT has been associated with resistance to chemoradiotherapy and recurrence of cancer (8).

Recently, it has been reported that the mesenchymal transition and EMT-activating transcription factors such as Snail family transcriptional repressor 1 (SNAI1), SNAI2 [previous HUGO gene nomenclature committee (HGNC) Symbols: Slug], and zinc finger E-box-binding homeobox 2 (ZEB2) induce aggressive behavior in aGB (9-16), suggesting that mesenchymal transition is one of the critical mechanisms in regulating aggressive behavior and the invasive properties of GBs.

Among several EMT-activating transcription factors, we focused on ZEB2, also known as Smad-interacting protein 1 (SIP1). ZEB2 was characterized for the first time from a Drosophila complementary DNA (cDNA) expression library (17, 18). ZEB2 expression was initially known to be closely related to aggressive clinicopathological features, including histological grade and overall survival in various epithelial tumors, such as ovarian carcinoma, gastric cancer, pancreatic cancer, and squamous cell carcinoma (10, 18-26). However, Xia et al. reported that ZEB2 is also highly expressed in aGB cells, and it regulates the migration and invasive ability of aGB cells (18).

In this study, we examined whether regulation of ZEB2 expression in both aGB and pGB cells affects mesenchymal transition. Also, the effect of ZEB2 gene regulation on the viability, proliferation, and growth cycle of aGB and pGB cells were assessed in vitro. Based on our results, this study provides novel insights into the differences between aGB and pGB in terms of the molecular mechanisms underlying tumor progression.

Materials and Methods

Cell culture. Human aGB cell lines, U87 and U373, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Japan Health Science Research Resources Bank kindly provided the human pGB cell line KNS42. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and antibiotics [penicillin (50 U/ml), and streptomycin (50 U/ml)] in a humidified 5% CO2 incubator at 37°C. The medium was replaced every 2-3 days.

Knockdown of ZEB2 expression. Small interfering RNAs (siRNA) targeting ZEB2 were used to inhibit endogenous ZEB2 in all cell lines. Two ZEB2-specific siRNAs and “scrambled siRNA (negative control)” were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and Bioneer (Daejeon, Republic of Korea), respectively. Details about the ZEB2 siRNAs targeting different regions of ZEB2 mRNA are provided in Table I. Routinely, cells were transfected with 25-50 nM siRNAs using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. Knockdown efficiency was measured at the mRNA level by quantitative RT-qPCR. The transfected cells were used for various assays such as Western blot, CCK-8 and BrdU assays, 48-72 h after transfection.

View this table:
Table I.

Sequences of the two ZEB2 specific siRNAs.

RNA extraction and RT-qPCR from fresh tissues and cell lines. Total RNA was extracted from transfected cells and powdered tissues using TRIzol reagent (Invitrogen) and PureLink RNA mini kit (Invitrogen) according to the manufacturer’s protocols. cDNA was subsequently synthesized using EcoDry Premix- Oligo (dT) (Clontech, Mountain View, CA, USA) from 1 μg of total RNA. Real-time PCR (Power SYBR® Green, ABI, Warrington, UK) analysis was performed using an ABI Prism 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, using gene-specific primers (Table II). Tumor samples were analyzed in triplicate, and gene expression levels were normalized against that of GAPDH.

View this table:
Table II.

Primer sequences for reverse transcription and quantitative real-time polymerase chain reaction.

Western blot. Total cell protein extracts were obtained by lysis in RIPA buffer (Thermo Scientific, Pittsburgh, PA, USA), and quantification was performed using the Pierce BCA® Protein Assay Kit (Thermo Scientific). The lysate was mixed with an equal volume of sample buffer, denatured by boiling, and then separated on NuPAGE sodium dodecyl sulfate (SDS) 4-12% gradient polyacrylamide gels (Invitrogen). The proteins were transferred to nitrocellulose membranes by using the iBlot dry transfer system (Invitrogen). Membranes were blocked with blocking solution for at least one hour at room temperature. Primary antibodies were incubated with the samples overnight at 4°C; ZEB2 (1:1,000; ProSci, Poway, CA, USA), E-cadherin (1:200; Abcam, Cambridge, UK), and vimentin (1:1,000; Cell Signaling, Danvers, MA, USA). The HRP-conjugated secondary antibody was then incubated with the samples for one hour at room temperature. The membranes were developed using the enhanced chemiluminescence detection system (Invitrogen) and visualized by exposure to an autoradiographic film (Kodak, Rochester, NY, USA).

Cell counting kit-8 (CCK-8) and BrdU assay. Cell viability was determined using CCK-8 (Dojindo Laboratories, Rockville, MD, USA). About 4×103 cells were seeded in 96-well culture plates and transfected with scrambled siRNA and ZEB2 siRNA. Following 24-72 h of incubation, CCK-8 reagent was added and incubated for two hours; then, the absorbance of each well was measured at 540 nm with a micro-ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

The proliferation of transfected cells was determined using a BrdU proliferation ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany). BrdU was added to the cells, the mixture was incubated for two hours, and the cells were treated according to the manufacturer’s instructions before measuring the optical density at 370 nm using an ELISA plate reader. The percentage of cell survival was determined based on the relative absorbance of cells transfected with ZEB2-specific siRNA versus cells transfected with scrambled siRNA. All assays were performed in triplicate.

FACS analysis. To assess cell cycle distribution, flow cytometry was performed. The cells were transfected with scrambled siRNA and ZEB2-specific siRNA. Three days after transfection, the cells were harvested, washed in phosphate-buffered saline (PBS), and fixed in 70% ethanol for 1 h at 4°C. Then, the cells were washed with PBS, rehydrated, and resuspended with 0.5 mg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA) in PBS buffer for 30 min at 37°C. The cells were stained with 10 μg/ml propidium iodide solution (Sigma-Aldrich) in the dark and analyzed for DNA content with a flow cytometer (FACS Caliber, BD, Heidelberg, Germany).

Invasion assay and wound healing assay. Cell motility and invasiveness were evaluated by wound healing assay and transwell migration assay. The wound-healing assay was performed with a CytoSelect™ 24-well cell invasion assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions and performed as described previously (15). Invasion of tumor cells was analyzed using a Cell Invasion Assay Kit (pore size, 8 μm; Chemicon, Burlington, MA, USA) according to the manufacturer’s protocol.

Immunohistochemistry. Immunohistochemical analysis was conducted by the automatic immunohistochemical staining equipment Lab vision Autostainer 480S (Lab Vision Corp., Fremont, CA, USA) according to the manufacturer’s protocol based on our previous reports (27). The primary antibody used in this study was ZEB2 (1:200 dilution; polyclonal; Abcam, Cambridge, UK). The positive control slide for ZEB2 was one from aGB cases that showed a higher level of ZEB2 mRNA and negative control specimen was obtained by omission of the primary antibody.

Patients. Eighteen patients with glial tumors (6 aGB, 8 pGB, 2 adult diffuse astrocytomas, 2 pediatric pilocytic astrocytomas) were included in the present study. Immunohistochemistry for ZEB2 protein was performed in samples from these patients. Fresh frozen tissues of aGBs (n=6), pGB (n=8), and non-neoplastic brain tissue (control brain tissue, n=2) were used for real–time polymerase chain reaction (PCR) analysis to detect ZEB2 mRNA.

Statistical analysis. All statistical data are presented as means±SDs, and GraphPad Prism software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. Statistical significance was determined by Student’s t-test. For comparison of more than three groups, we employed one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests. Differences were considered to be statistically significant when p<0.05.

Results

Down-regulation of ZEB2 expression reduced mesenchymal features in aGB and pGB cells. Inhibition of ZEB2 significantly increased the expression of E-cadherin at both mRNA and protein levels in aGB and pGB cells (Figure 1). In contrast, vimentin mRNA and protein levels were reduced in both aGB and pGB cells. Based on the changes of EMT markers after down-regulation of ZEB2 expression, we confirmed that mesenchymal features were reduced in both aGB and pGB cells.

Figure 1.
Figure 1.

Comparative analysis of mRNA and protein expression for EMT markers after ZEB2 siRNA and scrambled siRNA transfection in aGB (U373, U87) and pGB (KNS-42) cell lines. (A) Relative mRNA expression of ZEB2, E-cadherin, and vimentin after transfection of scrambled siRNA or two ZEB2-specific siRNAs. (B) Protein expression levels of ZEB2, E-cadherin, and vimentin after transfection of scrambled siRNA or two ZEB2-specific siRNAs. β-actin loading control is shown in the bottom panel.

Inhibition of ZEB2 reduced the invasive ability and migration of aGB and pGB cells. The results of invasion and wound healing assays demonstrated that inhibition of ZEB2 expression reduced the invasive ability of aGB and pGB cells (Figures 2 and 3). Treatment with ZEB2-specific siRNA also significantly reduced the migration ability of aGB and pGB cells.

Figure 2.
Figure 2.
Figure 2.

Invasion assay after ZEB2 siRNA and scrambled siRNA transfection in aGB and pGB cell lines. (A) Images displaying the cells that invaded through the filter pores after transfection of scrambled siRNA or two ZEB2-specific siRNAs. (B) Bar graph summarizing the average total number of invaded cells quantified using imaging software. *p<0.05, vs. scrambled siRNA (control).

Figure 3.
Figure 3.

Wound healing assay of aGB and pGB cell lines in response to ZEB2 siRNA or scrambled siRNA transfection. Cells were cultured and wound lines were introduced using a microtip. After incubation, the rate of gap closure was higher in cells transfected with scrambled siRNA compared to those transfected with ZEB2-specific siRNA. These results were common between pGB and aGB cells.

Inhibition of ZEB2 expression reduced proliferation and viability of pGB cells. Inhibition of ZEB2 expression significantly reduced proliferation and viability of pGB cells, while proliferation and viability of aGB cells were not affected (Figure 4). Therefore, down-regulation of ZEB2 reduced cell proliferation and viability only in pGB cells. Inhibition of ZEB2 gene expression induced G1-phase cell cycle arrest in pGB cells. Different distributions in the G1, S, and G2/M phases of the cell cycle were observed in both aGB and pGB cells. Cell cycle analysis demonstrated that inhibition of ZEB2 expression pGB cells significantly increased the proportion of cells in G1, while the proportion of cells in G2/S was reduced considerably. In contrast, aGB cells did not show significant changes in the proportion of cells in the G1 and G2/S phases (Figure 5). Similar to the results obtained in the proliferation and viability assays, inhibition of ZEB2 expression had different effects on pGB and aGB cells. These results indicate that ZEB2 down-regulation induced mesenchymal transitions in both pGB and aGB cells, but there was a differential effect on cell proliferation, viability, and cell cycle progression.

Figure 4.
Figure 4.

Cell proliferation and viability assays after ZEB2 siRNA or scrambled siRNA transfection in aGB and pGB cell lines (A) Line graph showing the effects of ZEB2 inhibition on the proliferation of aGB and pGB cell lines. Considerably decreased proliferation of cells after ZEB2 inhibition was only observed in pGB cells. *p<0.05, vs. scrambled siRNA (control). (B) Bar graph showing the effects of ZEB2 inhibition on the viability of aGB and pGB cell lines. Decreased cell viability was observed only in pGB cells after ZEB2 inhibition. *p<0.05, vs. scrambled siRNA (control).

Figure 5.
Figure 5.

FACS analysis after ZEB2 siRNA or scrambled siRNA transfection in aGB and pGB cell lines. (A) DNA content analysis by flow cytometry after propidium iodide staining revealed different cell cycle distributions in G1, S, and G2/M phases of cell cycle for aGB and pGB cell lines. Considerably increased proportions of cells in the G1 phase were observed only in pGB cells. Shown are representative FL2A histogram. (B) Quantification of cell cycle distributions (in percentage). *p<0.05 vs. scrambled siRNA (control).

Immunohistochemical study of ZEB2 in normal, pGB and aGB human patient samples. In normal brain parenchyma, astrocytes and neurons were weakly immunoreactive for ZEB2. In contrast, pGB and aGB tissue sections showed strong staining for ZEB2. There was no apparent difference in ZEB2 expression between pGB and aGB. Also, we evaluated the expression levels of ZEB2 in low-grade glioma (LGG) and GBs. Both adult and pediatric LGG were positive for ZEB2 along the nuclear membrane, but its staining intensity was fainter than that of pGB and aGB, and stronger than that of the normal astrocytes in normal brain tissue (Figure 6).

Figure 6.
Figure 6.

Immunocytochemical analysis of ZEB2 expression tumor tissues and cell lines. ZEB2 shows weak positive expression in astrocytes and neurons of normal brain tissue (A). Adult low-grade glioma cases (B, C) and pediatric low-grade glioma cases (D, E) show weak expression of ZEB2. High levels of ZEB2 expression are observed in aGB (F, G) and pGB tumor cells (H, I).

mRNA expression of ZEB2 in normal brain, pGB and aGB tissues of human patients. aGBs and pGB tissues of patients showed higher levels of ZEB2 mRNA expression than non-neoplastic brain tissues (mean±s.d. of ZEB2 mRNA/GAPDH mRNA: 2.689±1.351 in aGB; 1.030±0.3260 in pGB, 0.961±0.105 in normal brain tissue), but the difference in ZEB2 mRNA levels did not show statistical significance. The difference in the levels of ZEB2 mRNA expression between aGB and pGBs was statistically significant (p=0.008) (Figure 7).

Figure 7.
Figure 7.

Semiquantitative real time PCR analysis for ZEB2 mRNA expression in fresh-frozen normal brain tissues, adult glioblastoma tissues, and pediatric glioblastoma tissues. ZEB2 mRNA expression was significantly higher in aGB than pGB or normal brain tissues. ns: Not significant; *p<0.05.

Discussion

aGB and pGB are aggressive brain tumors; they display histological similarities, but their molecular pathogeneses remain unclear. Diverse molecular factors or mechanisms have been suggested to explain poor prognosis. One of them is EMT, which has been considered a crucial factor contributing to poor prognosis. Through EMT, cancer cells acquire diverse characteristics such as migration ability, treatment resistance, and stem cell properties. The tumor cells that acquire mesenchymal phenotype readily invade the surrounding tissues and show aggressive behavior (5, 6). Recently, the cancer stem cell theory has been proposed as a major cause of recurrence and resistance to therapy (8). Cancer cells harboring stem cell properties can survive after intensive treatment, giving rise to tumor recurrence and therapy failure. EMT induction enhances cancer stem cell properties. Therefore, understanding the role of mesenchymal transitions of aGB and pGB cells will help to develop optimal treatment strategies. Numerous studies have shown the critical role of the mesenchymal transition of cancer cells and EMT-activating transcription factors such as Snail, Slug, ZEB2 in oncogenesis.

In the present study, we studied the role of ZEB2 expression in GB cells, with emphasis on the differences between pGB and aGB cells. ZEB2 was initially identified as a Smad-interacting protein and is known to control the migration and invasion of cancer cells. The molecular hallmark of EMT is the loss of or decrease in E-cadherin expression and up-regulation of mesenchymal markers. ZEB2 is an EMT-activating transcription factor that functions through the regulation of E-cadherin expression. Furthermore, ZEB2 has been reported as an E-cadherin repressor and a primary regulator of tumor cell invasion (21-24). Therefore, we aimed to define EMT-like processes and the association between ZEB2 gene expression and tumor progression of pGB and aGB cells. Qi et al. have found that ZEB2 down-regulation resulted in the restoration of E-cadherin expression and suppression of vimentin expression in aGB cells, including U87 and U251 glioma cell lines (10). Consequently, we examined whether inhibition of ZEB2 expression would have the same effect on E-cadherin and vimentin expression and indeed we confirmed this at both mRNA and protein levels. We further investigated whether the changes in EMT markers encompassed phenotypic changes of glioma cells. To assess this, we compared the migration and invasive ability of aGB and pGB cells after siRNA-mediated ZEB2 knockdown. Inhibition of ZEB2 gene expression significantly reduced the invasive and migration abilities of both cell lines. Based on these results, we conclude that pGB and aGB cells underwent a mesenchymal transition at both molecular and phenotypic levels.

Overexpression of transcription factors of the ZEB family, including ZEB1 and ZEB2, have been shown to be correlated with cancer stem cell phenotype and treatment resistance in epithelial cancers (28, 29). ZEB1 expression, has been shown to be associated with the acquisition of a highly infiltrative phenotype by aGB and pGB cells in hypoxic conditions (30-32). In pGB, characterized by PDGFRα overexpression and activation, the ZEB1-PDGFRα axis has been shown to be essential for mesenchymal transition (31, 33, 34). Unlike the role of ZEB1 expression in pGB, ZEB2 expression has been correlated with higher tumor grade and mesenchymal properties in aGB. Therefore, the information regarding the role of ZEB2 in pGB was limited. In terms of cell proliferation, viability, and cell cycle distribution, our study revealed differences between pGB and aGB cells after ZEB2 gene inhibition. ZEB2 knockdown by siRNA resulted in significantly reduced pGB cell proliferation and viability and an increased cell population at the G1 phase of the cell cycle. These results, however, were not observed in aGB cells.

In contrast, ZEB2 gene inhibition in aGB cells had no effect on cell proliferation, viability or cell cycle distribution. In aGB cells, the proportion of cells in the G1 phase did not increase after ZEB2 down-regulation. Also, these results are different from those obtained by Qi et al. (10). They provided experimental evidence for ZEB2-regulated cell cycle progression, migration, invasion, and apoptosis in U251 and U87 glioma cells. Although the effects on invasion and migration are comparable with our results, the influence of ZEB2 inhibition on cell cycle progression is different. Our analyses revealed significant effects on cell cycle progression only in pGB cells, not in aGB cells.

In contrast to Qi et al., we found that ZEB2 inhibition did not affect cell proliferation or viability of U87 and U373 aGB cell lines. Thus, we concluded that although ZEB2 down-regulation induced significant reduction of EMT-like properties in both aGB and pGB cells, ZEB2-dependent effects on cell proliferation, viability, and growth cycle distribution were apparent only in pGB, but not in aGB, cells. One of the reasons for this difference may be that different downstream molecules and/or genetic factors underlie ZEB2 gene regulation in aGB and pGB cells, explaining the different pathogenetic mechanisms in these two types of tumors. Also, we performed immunohistochemical staining of ZEB2 on formalin-fixed paraffin-embedded tissues, including GB, low-grade gliomas, and normal brain from both pediatric and adult cohorts. As a result, normal astrocytes and low-grade glioma cells showed very weak ZEB2 expression; however, strong staining patterns were observed for aGB and pGB. Although no visible differences in ZEB2 protein expression between aGB and pGB of the same WHO grade were observed by immunohistochemistry, significant difference in ZEB2 mRNA expression was observed between aGB and pGB compared to normal fresh frozen brain tissues, aGB and pGB also showed higher ZEB2 mRNA expression. Furthermore, distinct differences were observed between low-grade and high-grade gliomas. Corroborating our results, Qi et al. have found that ZEB2 protein expression is associated with the status of WHO grade. These results indicate the role of ZEB2 in glial tumors. High-grade gliomas typically demonstrate more invasive properties and greater resistance to treatment than low-grade gliomas, which is similar to the characteristics observed in many epithelial cancers following EMT induction. It is possible that high-grade glioma cells acquired these properties by mesenchymal transition through the up-regulation of ZEB2 expression, and that these features had differential effects on pGB and aGB cells.

In conclusion, we found that ZEB2 expression fulfills an oncogenic function in both pGB and aGB cells, but pGB cell proliferation, viability, and cell cycle progression were affected more by ZEB2. These differences suggest that different underlying molecular and genetic mechanisms affect tumor progression in pGB and aGB. Our results indicate that ZEB2 expression could be used as diagnostic and prognostic marker, and therapeutic target. The inhibition

of EMT could be one of the therapeutic strategies for highly aggressive GB, but pGB and aGB should be approached differently.

Acknowledgements

This work was supported by the research fund of Hanyang University (HY-202000000002704) and Seoul National University (0320110210).

Footnotes

  • Authors’ Contribution

    Conceptualization, Jae Kyung Myung and Sung-Hye Park; Formal analysis and Validation, Jae Kyung Myung, Seung Ah Choi, and Sung-Hye Park; Methodology, Jae Kyung Myung, Seung Ah Choi; Funding acquisition, Jae Kyung Myung and Sung-Hye Park; Supervision, Seung-Ki Kim, Sung-Hye Park; Writing – original draft, Jae Kyung Myung; Writing – review & editing, Seong Ik Kim, Jin Woo Park, Sung-Hye Park; All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors do not have any conflicts of interest to declare regarding this study.

  • Received November 5, 2020.
  • Revision received November 20, 2020.
  • Accepted November 21, 2020.

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The Role of ZEB2 Expression in Pediatric and Adult Glioblastomas
JAE KYUNG MYUNG, SEUNG AH CHOI, SEUNG-KI KIM, SEONG IK KIM, JIN WOO PARK, SUNG-HYE PARK
Anticancer Research Jan 2021, 41 (1) 175-185; DOI: 10.21873/anticanres.14763
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