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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 221-227

Tumour suppressive effects of WEE1 gene silencing in neuroblastomas


1 Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Immunology, Immunotherapy Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Web Publication13-Apr-2016

Correspondence Address:
Mojtaba Habibagahi
Department of Immunology, Immunotherapy Laboratory, School of Medicine, Shiraz University of Medical Sciences, Zand Boulevard, Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.165861

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 > Abstract 


Aim of Study: WEE1, a member of serine/threonine protein kinase family is the master inhibitor of cyclin-dependent kinase 1 in cell cycle. Over-expression of WEE1 in glioblastomas (GBMs) and some other cancers has been shown. Here, we investigated the expression of WEE1 in 13 brain samples from GBM patients and two GBM cell lines. Further to that, we asked whether if knocking down WEE1 expression in the cell lines change tumor cells' reaction.
Materials and Methods: All brain tumor samples were collected after confirmed pathological diagnosis. Western blotting was used to screen the expression of WEE1 and a panel of tumor markers. As a model of WEE1 gene silencing with small hairpin RNA (shRNA) technology in GBMs, A172, and U373GM cell lines were transfected with four WEE1 specific shRNAs. The growth characteristics of the cells and the expression of a panel of downstream genes were investigated after gene suppression.
Results: All GBMs and both cell lines over-expressed WEE1. Transduction of the cell lines with different shRNAs suppressed WEE1 expression with different extent and pooling of four shRNAs together resulted in additive effect. Suppression of WEE1 not only repressed cellular growth but also changed the profile of gene expression of the cells. Quantitative real-time polymerase chain reaction showed also reduced expression of genes such as hypoxia-inducible factor-1, B-cell lymphoma-2, vascular endothelial growth factor, and p53 with crucial roles in tumor survival and invasiveness.
Conclusion: These results highlight the key role of WEE1 suppression to combat GBMs. Moreover, it showed beneficial possibilities of WEE1 suppression with different anticancer approaches for neurological malignancies.

Keywords: Gene suppression, glioblastoma, neuroblastoma, small hairpin RNA, WEE1


How to cite this article:
Tashnizi AH, Jaberipour M, Razmkhah M, Rahnama S, Habibagahi M. Tumour suppressive effects of WEE1 gene silencing in neuroblastomas. J Can Res Ther 2016;12:221-7

How to cite this URL:
Tashnizi AH, Jaberipour M, Razmkhah M, Rahnama S, Habibagahi M. Tumour suppressive effects of WEE1 gene silencing in neuroblastomas. J Can Res Ther [serial online] 2016 [cited 2021 Jan 24];12:221-7. Available from: https://www.cancerjournal.net/text.asp?2016/12/1/221/165861




 > Introduction Top


Glioblastoma (GBM) is one of the most aggressive brain tumors with a median survival of 15 months.[1] There have been great efforts to help the patients; however, surgery, radiation, and chemotherapy techniques have not made major improvement in clinical outcome of the patients.[2] Studies showed that any success in anticancer therapy should reach two goals of growth inhibition and induction of cell death.[3] However, GBM cells are far resistant to apoptotic cell death.[4] This fact accentuates the need for novel therapy approaches to win the battle against GB. In a normal cell, DNA-damages induce guardian checkpoints of cell cycle to activate a series of DNA repair mechanisms at G1-S transition, S-phase, and G2-M transition. In some cancer cells however, because of mutation in p53, the G1-checkpoint is defective and cells are more dependent on the G2 checkpoint to repair DNA-damages.[5],[6] It means, abrogation of the G2 checkpoint in such cancer cells could arrest DNA repair, induce premature mitotic entry, and initiate apoptosis. Therefore, it is possible to exploit the G2 checkpoint as a potential target for cancer therapy in some types of cancers.[7]

WEE1 is a nuclear protein, member of tyrosine kinase family which plays a major role in cell-cycle and G2-M transition.[8] During normal circumstances, it plays a major role in controlling the timing of mitosis. Activated WEE1 causes inhibitory phosphorylation of CDC2/cyclin-dependent kinase 1 on Tyr15, which prevents cells to enter mitosis in order to provide extra time for DNA repair.[9] Results have indicated WEE1 over-expression in many human cancers.[10] Inhibition of WEE1 with siRNA showed selective killing of cancer cells with DNA-damage and promoted replication stress at G2 checkpoint.[9] Similarly, inhibition of WEE1 could improve the DNA-damage effect after radiotherapy and chemotherapy in different cancers.[11],[12]

Therefore in this study, we screened the expression levels of WEE1 in the tissue samples from GBM patients and two GBM cell lines. We also investigated cellular growth characteristics and the expression of a panel of genes for angiogenesis, apoptosis, and hypoxia in two GBM cell lines, A172 and U373GM, after WEE1 was suppressed with gene-specific small hairpin RNAs (shRNAs).


 > Materials and Methods Top


Tissue samples

Fresh frozen GBM specimens (n = 13, average age = 45) and nonneoplastic epilepsy specimens as control (n = 4) were provided by Neurosurgery Department of Nemazi Hospital, Shiraz, Iran. Samples were diagnosed by histological examination and categorized according to clinical criteria. The study protocol and the experiments working on materials derived from human tissue were reviewed and approved by the Ethics Committee of Shiraz University of Medical Sciences.

Cell lines and cell culture

Two human GBMs cell lines, A172, and U373MG were purchased from the National Cell Bank at the Pasteur Institute, Tehran, Iran. The cell lines were cultured in RPMI-1640 medium (Sigma-Aldrich, Hong Kong, China) supplemented with 10% fetal bovine serum, 100 unit/ml penicillin, and 100 µg/ml streptomycin. Cells were grown in standard culture conditions and sub-cultured at 70–80% confluency.

WEE1 small hairpin RNA plasmids transfection

Plasmids transcribing shRNAs against WEE1 were purchased from Origene Company (Beijing, China). The sequence of four 29mer shRNA and the target locations of binding are listed in [Table 1]. Lipofectamine 2000 (Invitrogen, Grand Island, New York, USA) was used to transfect A172 and U373MG cell lines. To this end, 5 × 105 cells were transfected with 4 µg of single or pooled mixture (1 µg of each) of shRNA plasmids according to the manufacturer's instruction. A mixture of Scrambled shRNA was included in all experiments and used as a control.
Table 1: The sequence of each 29mer shRNA construct and its target location

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Quantitative real-time polymerase chain reaction

Quantification of gene expression at mRNA level was performed using quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from the transfected cells using Trizol reagent 24 h posttransfection. Reverse transcription to cDNA was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Helsinki, Finland). Real-time PCR was carried out to determine the expression levels of target genes using specifically designed primers and probes [Table 2]. Each sample was normalized against β-actin expression. The expression levels of target genes were determined by ΔCt and 2−ΔCt formulas.
Table 2: Sequences of primers and probes used in the real-time PCR experiment

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Western blotting

Western blotting was used to assess the expression of WEE1, hypoxia-inducible factor (HIF), vascular endothelial growth factor (VEGF), p53 and B cell lymphoma-2 (Bcl-2) in tumor biopsies or cell lines. To do that, tissue samples or the cell lines were homogenized by radioimmunoprecipitation assay buffer contained protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich). Cell lysates were subjected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (BioRad, Hercules, California, USA) and blocked. Membranes were probed with rabbit primary antibodies against β-actin (as an internal control), WEE1, HIF, VEGF, p53, and Bcl-2. Horseradish peroxidase conjugated goat polyclonal antibody against rabbit IgG-H, and L was used to reveal the bands on the Kodak XAR film with ECL detection reagents (Thermo SIENTIFIC, Waltham, Massachusetts, USA). All primary and secondary antibodies were purchased from Abcam, UK.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The cytotoxic effect of WEE1 silencing in the glioblastoma cell lines was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assays after 12, 24, 48, 72, and 96°h inhibition with the gene specific shRNAs. To this end, cells (15 × 103) were seeded into wells of 96 well plates for an overnight culture in triplicates and transfected with WEE1 shRNAs using lipofectamine. Afterward, the media was replaced by MTT solution (Sigma, USA) and processed as per directed by the manufacturer. The absorbance of color formation in the wells was read at 490°nm. The following equation was used to calculate the percentages of viable cells in each well:

(Viable cells)% = (OD490 transfected/OD490 untreated) ×100

Statistical analysis

Differences between data were analyzed using Mann–Whitney U-test. P values smaller than 0.05 was considered as statistically significant.


 > Results Top


Molecular analysis of glioblastoma samples

[Figure 1] shows typical examples of the Western blotting analysis on GBM specimens in comparison to control samples. Clear increased expression of WEE1, HIF-1, VEGF, Bcl-2, and p53 in the GBM samples was observed. Similarly, mutant p53 showed stronger expression in the tumor samples.
Figure 1: Typical example of Western blotting analysis for the expression of the target markers. Expression of WEE1, hypoxia-inducible factor-1, vascular endothelial growth factor, B-cell lymphoma-2 and p53 by glioblastoma specimens and nonneoplastic epilepsy specimens were studied by Western blotting. In all experiments, expression of β-actin was used as internal control. Stronger expression of the target markers in all glioblastoma samples was evident

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WEE1 gene silencing in glioblastoma cell lines

In order to investigate the effect of WEE1 inhibition in A172 and U373MG glioblastoma cell lines at the molecular level, cells were transfected with single or pool of four WEE1 shRNAs. At 48 h posttransfection, Western blotting results from both cell lines showed a major decrease of WEE1 expression in the cells transfected with the pool of four shRNAs [Figure 2]. The expression level of phosphorylated CDC2 which can evaluate the efficiency of WEE1 silencing was reduced in both GBM cell lines after the transfection too. [Figure 2]a and [Figure 2]b demonstrate typical examples of reduced expression of the target markers in compared to β-actin and controls in A172 and U373MG cell lines, respectively. When the expression of WEE1 was assessed at the transcriptional level by qRT-PCR, decrease in gene expression could be detected at earlier time points. At 24 h posttransfection, the relative levels of WEE1 transcripts in A172 cells showed 10%, 40%, 65%, 45%, and 70% reduction of the control cultures when the cells were transfected with WEE1 shRNA 1, 2, 3, 4, and pool of four shRNAs, respectively [Figure 2]c. As shown in [Figure 2]d, at the same time point, the expression level of WEE1 in U373MG cells had up to 4%, 43%, 50%, 63%, and 98% reduction compared to the controls when WEE1 was silenced with shRNA 1, 2, 3, 4, or pool of all, respectively [Figure 2]d.
Figure 2: WEE1 expression by A172 and U373MG cell lines. Gene expression profiles of A172 and U373MG cells were studied by Western blotting (a and b) and quantitative real-time polymerase chain reaction (c and d) 48 h after cells were transfected with different shRNA constructs encoding WEE1 specific sequences or the mixture of scrambled shRNAs as control. Expressions of β-actin were used as internal control for Western blot analyses. Real-time polymerase chain reaction results were also normalized against β-actin expression. Results showed down-regulation of WEE1 in the cells after the transfection where additive effect of pooled transfection was evident. Down-regulation of phosphorylated-DC2 could also confirm the suppression of WEE1 too

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium proliferation assay

The effects of WEE1 silencing on the viability and growth capability of GBM cell lines were assessed by MTT assay. The assays were performed at different time point up to 96°h after the transfection. Significant inhibition of A172 and U373MG cellular growth/loss of viability by WEE1 gene silencing was observed 48 h posttransfection [Figure 3]a and [Figure 3]b which was used for further analysis (P = 0.0079 and 0.03, respectively). As it was shown in [Figure 3], greatest suppression of cell viability (up to 60%) was achieved after the wee1 gene was silenced with a pool of four shRNAs. Compared to U373MG cells, relatively less growth inhibition was demonstrated in A172 cells after WEE1 silencing.
Figure 3: Cell viability of A172 and U373MG after WEE1 suppression. Cell viability of A172 cells (a) and U373MG cells (b) were measured 48 h after the cells were transfected with shRNA 1, 2, 3, 4, pool of four constructs or a mixture of scrambled small hairpin RNA as control by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. Viability of Compared to U373MG cell line, A172 cells were slightly less affected by WEE1 suppression with single small hairpin RNA transfection, however, greater loss in cell viability happened after WEE1 was suppressed with pool of four small hairpin RNAs

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Change of molecular profile in the cell lines after WEE1 inhibition

To investigate the relation of WEE1 and some downstream genes, the molecular expression profile of HIF-1, VEGF, Bcl-2, and tumor protein p53 were evaluated in A172 and U373MG cell lines after WEE1 gene was silenced with gene specific shRNAs [Figure 4].
Figure 4: Expression measurement of selected genes downstream of WEE1. Quantitative real-time polymerase chain reaction analysis was used to measure relative expression levels of hypoxia-inducible factor-1, vascular endothelial growth factor, B-cell lymphoma-2 and p53 genes 24 h after WEE1 was suppressed by four gene-specific small hairpin RNA, pool of four or scramble control in A172 (a, c, e and g) and U373MG (b, d, f, and h) cells. WEE1 suppression resulted in down-regulation of the genes by different extent. WEE1 suppression by a mixture of four small hairpin RNAs resulted in greater down-regulation of the studied genes

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A significant decrease in the expression of HIF-1 was apparent 24 h after WEE1 was inhibited in the cells. In A172 cells, the expression of HIF-1 transcripts showed moderate suppression with shRNA 1 and 2 (up to 40%, P < 0.029). Highest reduction in transcription (up to 80%) happened when cells were transfected with shRNA 4 (P = 0.03), or it was fully suppressed with a pool of four shRNAs [Figure 4]a. Similarly, HIF-1 expression reduced in U373MG cells after WEE1 silencing. shRNA 1 the least effect (P = 0.07) however, others could suppress HIF-1 expression up to 75% (P = 0.02) and mixture of all four shRNAs almost fully suppressed the expression of HIF-1 when compared to the controls [Figure 4]b. Examination of VEGF also showed a significant decrease after 24°h transfection of the cells with WEE1 shRNAs. In A172 cell lines, the VEGF transcripts showed reduction between 20% and 50% compared to the controls, when WEE1 was silenced with either of shRNAs, except shRNA 1. The combination of four shRNAs together could bring down the expression of VEGF in A172 cells up to 80% of the controls [Figure 4]c, P = 0.04]. In U373MG cells, WEE1 silencing with shRNAs 1 and 2 had the only moderate effect to down-regulate the VEGF expression. However, both shRNA 3 and shRNA 4 suppressed major VEGF expression (up to 85% of the controls, P = 0.03). Full suppression of the expression was achieved when the cell line was transfected with the mixture of shRNAs [Figure 4]d. Bcl-2 gene expression also decreased in the cell lines 24 h after WEE1 gene silencing. At this time point shRNAs 2, 3 and 4 were more effective and could suppress Bcl-2 up to 70% compare to the controls (P < 0.05). Combination of all shRNAs however did not augmented Bcl-2 suppression in A172 cells [Figure 4]e. U373MG cells showed similar range of reduction in Bcl-2 expression after WEE1 silencing with shRNA 2, 3, 4, and mixture of four (P < 0.05). However, suppression with shRNA 1 did not change the expression of Bcl-2 in U373MG cells [Figure 4]f. The expression of p53, the key regulator of G1-checkpoint, barely affected after WEE1 suppression in some experiments. Using shRNA 1 and shRNA 4 to suppress WEE1 in A172 cells, had only minor effect on p53 expression (P > 0.1). Silencing of WEE1 with shRNA 2 and shRNA 3 decreased the levels of p53 transcripts up to 50% of the controls; however, it was not statistically significant (P = 0.07). When WEE1 was suppressed in the cells with the combination of all shRNAs, a moderate suppression of p53 expression, up to 30%, was demonstrated [Figure 4]g. U373MG cells also moderately decreased the expression of p53 after WEE1 was suppressed. The decrease was more uniform with different shRNAs in U373MG cells and reached to 42% with a pool of four shRNAs compared to the controls [Figure 4]h. However, the reduction was not statistically significant (P = 0.067).


 > Discussion Top


Here, we demonstrated the over-expression of WEE1 in GBM brain tissue samples and two established GBM cell lines. Both in silico analyses and experimental results have demonstrated the over-expression of WEE1 in GBMs.[13],[14] Literature shows the over-expression of WEE1 is not restricted to neurological malignancies as other tumors such as ovarian cancers and melanomas over-expressed this cell cycle regulator. Interestingly, in many cases the expression of WEE1 was in association with poor survival of the patients.[15],[16]

Different substances such as AZD1775, MK1775, or microRNA-497 have been used to inhibit WEE1 expression in different studies.[17],[18],[19] In our hand, transient transfections of gene specific shRNAs, targeting four different sequences in WEE1 transcript could also suppress WEE1 expression in A172 and U373MG cell lines. Neither of the single shRNAs that we used could fully suppress the expression of WEE1 and the magnitudes of suppression varied cell to cell with different shRNA variants. However, a combination of all shRNAs in pooled transfection experiments resulted in major gene knockdown in both cell lines after 48 h. In spite that even partial suppression of WEE1 by single shRNAs could affect the survival of the GBM cell lines. The combination of four shRNAs in A172 cells had an additive effect to inhibit a larger fraction of the cells and stopped almost all the cultured cells. However, pooled mixture of shRNAs did not significantly increase growth suppression of U373GM cells which show the possible difference of downstream pathways in different target cells. Suppression of WEE1 could affect several downstream genes in A172 and U373GM cells like HIF-1. The HIF-1 plays a crucial role in responses to hypoxia. The association of HIF-1 overexpression and brain tumor development/progression has been demonstrated.[20] We demonstrated a significant reduction of HIF-1 expression after WEE1 silencing. To the best of our knowledge, there are no previous reports showing down-regulation of HIF-1 by inhibition of WEE1 expression. Interestingly, hypoxia could also induce WEE1 over-expression where it protected the cells against hypoxia damages.[21] It means WEE1 suppression may provide a dual benefit to inhibit the growth of neurological cancer cells. VEGF, a gene downstream of HIF-1, also was down-regulated after WEE1 was suppressed. The VEGF and its receptors also play a critical role in the development of tumor vasculature.[22] Increased VEGF expression has been found in association with GBM aggressiveness and poor prognosis.[23] Therefore, inhibition of WEE1 not only can abrogate G2-arrest rescue pathway in cancer cells and induce mitotic catastrophe but also by down-regulation of HIF-1 and VEGF expression can reduce the survival of cancer cells during hypoxia. p53 is the key regulator of the G1-checkpoint. In our results, primary GBM samples showed increased expression of p53 which is in contrary to most but not all results.[24],[25] WEE1 suppression had minimal effect on p53 expression, and it varied in the two cell lines. In spite that our previous study showed a significant increase of p53 after WEE1 inhibition in MCF-7 breast cancer cell line [26] which highlights great variations regarding the dependency of p53 to WEE1 in different cells. Although GBM cell lines with wild-type p53 were able to respond to radiation, the literature shows that most GBMs are p53 deficient and carry the mutant form of the gene. Specific inhibition of WEE1 in cells with inactive p53 could inhibit cellular proliferation.[6] We also demonstrated a significant decrease in cell survival of the cells after WEE1 suppression. A172 and U373GM like many other GBMs showed high expression of Bcl-2, which makes cells more resistance to therapies.[27] WEE1 suppression, however, reduced the level of Bcl-2 expression in the cells which would be advantageous for cancer therapies. All these data shows the major additive effect of WEE1 suppression for neurological cancer therapies. In fact, this has been the basis of some recent experimental therapies to apply WEE1 inhibition with radiation-induced changes in GBMs for more successful therapy.[28]


 > Conclusion Top


Results support beneficial effects of WEE1 suppression in conjunctional therapies for different cancers and, in particular, neurological cancers. It not only targets pathways directly controlled by WEE1 but also makes cancer cells more vulnerable to anticancer agents by suppressing essential mechanisms such as angiogenesis. Cancers are highly variable, therefore, more confirmatory results should be provided to find the ideal target and means of suppression for successful cancer therapy.

Acknowledgments

This study was financially supported by a grant from Shiraz University of Medical Sciences. Some of the results presented in this article were extracted from the thesis of S. Rahnama submitted to the Shiraz University of Medical Sciences for Degree of Master of Science.

Financial support and sponsorship

This work was supported by grants from Research deputy of Shiraz University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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