Research ArticleExperimental Studies

CEP-1347 Targets MDM4 Protein Expression to Activate p53 and Inhibit the Growth of Glioma Cells

YUTA MITOBE, YURIKA NAKAGAWA-SAITO, KEITA TOGASHI, SHUHEI SUZUKI, ASUKA SUGAI, KEN-ICHIRO MATSUDA, YUKIHIKO SONODA, CHIFUMI KITANAKA and MASASHI OKADA
Anticancer Research October 2022, 42 (10) 4727-4733; DOI: https://doi.org/10.21873/anticanres.15977

Abstract

Background/Aim: The development of pharmacological inhibitors targeting negative regulators of p53, such as murine double minute (MDM) 2 and, more recently, MDM4, has been actively pursued as a potential strategy to treat cancers with wild-type p53. We previously showed that CEP-1347, a small molecule kinase inhibitor originally developed for the treatment of Parkinson’s disease, suppressed MDM4 expression and activated wild-type p53 in retinoblastoma cells. However, it remains unknown whether CEP-1347 acts as an MDM4 inhibitor and as such activates p53 in other types of human cancer cells. Materials and Methods: The effects of CEP-1347 and MDM4 knockdown on the mRNA and protein expression of components of the p53 pathway, including MDM4, in human glioma cell lines with and without p53 mutation were examined by RT-PCR and western blot analyses. Trypan blue dye exclusion was used to examine the effect of CEP-1347 on cell growth. Results: CEP-1347 decreased the expression of MDM4, increase that of p53, and activated the p53 pathway in glioma cells with wild-type p53. Knockdown-mediated inhibition of MDM4 expression in a glioma cell line with wild-type p53 that overexpresses MDM4 resulted in increased p53 expression and activation of the p53 pathway. CEP-1347 preferentially inhibited the growth of glioma cells with wild-type p53 without showing toxicity to normal cells at clinically relevant concentrations. Conclusion: Our findings suggest CEP-1347 is a novel inhibitor of MDM4 protein expression and as such activates p53 to inhibit the growth of cancer cells with wild-type p53, including retinoblastoma and glioblastoma.

Key Words:

The p53 protein, encoded by the TP53 gene, is a transcription factor that orchestrates the expression of genes implicated in such cellular processes, but not limited to, as cell cycle, apoptosis, senescence, and DNA repair, that help prevent cells under various stresses from undergoing genomic alteration and neoplastic transformation. Thus, in normal cells, p53 functions through regulation of those target genes as “the guardian of the genome” and, as such, is inactivated in most, if not all, human cancers. In approximately half of human cancer cases, p53 is inactivated genetically by gene mutation and/or deletion and, in the remainder, p53 inactivation occurs functionally, for instance, through aberrant activation of its negative regulators represented by murine double minute (MDM) 2 and MDM4. Targeting such negative regulators of p53 deregulated in human cancers with wild-type p53 is, therefore, deemed a potential strategy to therapeutically reactivate p53 and has been drawing considerable attention (1-4).

CEP-1347 is a small molecule inhibitor of mixed lineage kinase originally developed for the purpose of promoting neuronal survival (5, 6). Although CEP-1347 failed to demonstrate its efficacy in treating early Parkinson’s disease in a large-scale phase 2/3 trial (7), accumulating evidence by our group and others has shed light on the pleiotropic anti-cancer activities of this drug, including its abilities to target drug resistance and cancer stem cells (8-14). Of note, we recently reported that CEP-1347 inhibited the growth of MDM4-overexpressing retinoblastoma cells with wild-type p53 in a p53-dependent manner (15). Moreover, the results of the study demonstrated that CEP-1347 reduced expression levels of MDM4 and increased those of p53 in retinoblastoma cells, which led to the intriguing possibility that CEP-1347 exerts its growth inhibitory effect by mitigating the negative impact of MDM4 on p53 expression. However, this possibility has yet to be tested experimentally due to the low transfectability of cultured retinoblastoma cells and remains an open question. More importantly, it also remains unknown whether CEP-1347 exhibits a similar, p53-dependent anti-cancer activity toward other types of human cancer cells in addition to retinoblastoma cells. Herein, we addressed these questions using human glioma cells with and without p53 mutation.

Materials and Methods

Reagents and antibodies. CEP-1347 was purchased from TOCRIS Bioscience (Bristol, UK) and was dissolved in DMSO to prepare a 0.5 mM stock solution. Trypan blue solution (T8154) was purchased form Merck (Darmstadt, Germany). An antibody (BL-3-2F2) against murine double minute 4 (MDM4) was purchased from BETHYL (Fortis Life Sciences, Waltham, MA, USA). An antibody against MDM2 (AF1244) was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against cyclin dependent kinase inhibitor 1A (CDKN1A, p21Waf1/Cip1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). An antibody against p53 (sc-126) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cell culture. The U-87 MG human glioblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The A172 and T98G human glioblastoma cell lines were provided by the RIKEN RBC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan (Tsukuba, Japan). The IMR90 normal human fetal lung fibroblasts were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) Cell Bank. U87 and IMR90 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA). A172 and T98G cells were maintained in RPMI1640 medium supplemented with 10% FBS. The culture media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. All IMR90 experiments were performed using cells with a low passage number (<8).

Western blot analysis. Western blot analysis was conducted as previously described (16, 17). Cells were harvested and washed with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer [10 mM Tris/HCl (pH 7.4), 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1.5 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail set III (FUJIFILM Wako Chemicals, Osaka, Japan)]. The lysates were immediately mixed with the same volume of 2× Laemmli buffer [125 mM Tris/HCl (pH 6.8), 4% SDS, and 10% glycerol] and boiled at 95°C for 10 min. After protein concentration of the cell lysates was measured using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA), samples containing equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were probed with the indicated primary antibodies followed by appropriate HRP-conjugated secondary antibodies as recommended by the manufacturer of each antibody. Immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and detected by a ChemiDoc Touch device (Bio-Rad).

Reverse transcription-PCR analysis. Reverse transcription (RT)-PCR analysis was conducted as previously described (17). Total RNA was extracted from cells using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μg of total RNA was reverse transcribed using the PrimeScript RT reagent kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. Target genes were amplified with Quick Taq HS DyeMix (Toyobo CO., LTD., Osaka, Japan). The sequences of gene-specific primer sets are listed in Table I.

View this table:
Table I.

Sequences of PCR primers.

Trypan blue dye exclusion assay. The numbers of viable and dead cells were determined using the trypan blue dye exclusion assay (18). Both adherent and non-adherent cells in culture dishes were collected, and after centrifugation, resuspended in PBS and stained with 0.2% trypan blue for 1 min. Viable and dead cells were identified by their ability and inability, respectively, to exclude trypan blue using a hemocytometer.

Gene silencing by siRNA. siRNAs against human MDM4 (#1: HSS106417, #2: HSS106418, and #3: HSS106419) and medium GC duplex #2 of Stealth RNAi siRNA negative control duplexes were purchased from Thermo Fisher Scientific. Cells were transiently transfected with one of the siRNAs against MDM4 (siMDM4; 160-200 pmol per 6-cm dish) or with the control siRNA (siCt; 160-200 pmol per 6-cm dish) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Statistical analysis. Results are shown as mean±standard deviation (SD). Data were analyzed using the Student’s t-test for comparisons between two groups. Differences with a p-value <0.05 were considered to be significant and are indicated with asterisks in the figures.

Results

p53 pathway status in glioma cell lines with and without p53 mutation. Prior to examining whether and how CEP-1347 exerts a p53-dependent anti-cancer activity on glioma cells, we first determined the status of the p53 pathway in cell lines to be used in this study under normal (unstimulated) culture conditions (Figure 1). Consistent with previous reports, whereas p53 mRNA was expressed at similar levels, the baseline p53 protein level was remarkably higher in T98G glioma cells expressing a mutant p53 protein than in A172 and U87 glioma cells expressing wild-type p53, most likely because mutant p53 proteins escape ubiquitin-dependent degradation (19-22). The mRNA and protein expression levels of p53 target genes, such as CDKN1A (p21) and MDM2 were in general higher in A172 and U87 compared to T98G cells. Consistent with a previous report demonstrating that the MDM4 gene was expressed in all gliomas irrespective of the presence or absence of gene amplification (23), which occurs in more than 4% of glioblastoma cases (23, 24), all three glioma cell lines expressed detectable levels of the MDM4 protein. Interestingly, the MDM4 protein level in A172 was remarkably high compared to its MDM4 mRNA level, suggesting that some post-transcriptional regulation of MDM4 expression may exist in glioma cells, as well as in other types of cancer cells (25-27).

Figure 1.
Figure 1.

The status of the p53 pathway in glioma cells with wild-type (A172, U87) and mutated (T98G) p53. The indicated cells maintained in their respective culture conditions without any stimulation were subjected to western blot (A) and quantitative RT-PCR (B) analyses for the expression of murine double minute (MDM) 4, MDM2, p53, and cyclin dependent kinase inhibitor 1A (CDKN1A, p21Waf1/Cip1).

CEP-1347 reduces the expression of MDM4 and activates the p53 pathway in glioma cells with wild-type p53 but not in glioma cells with mutated p53. We then examined the impact of CEP-1347 treatment on the expression of MDM4 and p53. Since the results of earlier clinical studies indicated that the blood concentration of CEP-1347 can reach as high as 700 nM safely in humans (28), we chose 500 nM as a clinically relevant concentration of CEP-1347 and treated cells with CEP-1347 at this concentration. Just as we observed in retinoblastoma cells (15), treatment of A172 and U87 cells with CEP-1347 resulted in a substantial reduction in MDM4 protein expression accompanied by a reciprocal increase in the protein but not mRNA levels of p53 (Figure 2). Importantly, the expression of p53 targets CDKN1A (p21) and MDM2 was increased both at the mRNA and protein levels along with p53 after treatment with CEP-1347 (Figure 2), underscoring that the increased p53 protein expression led to enhanced transcriptional activation of its target genes. Notably, CEP-1347 did not affect the mRNA levels of MDM4 in A172 and U87 cells. Furthermore, neither the mRNA nor the protein level of MDM4 decreased when T98G cells were treated with CEP-1347 (Figure 2). These findings may imply that CEP-1347 targets some post-transcriptional mechanism of MDM4 expression specifically active in glioma cells with wild-type p53. Put together, the results indicated that CEP-1347 reduced the expression of MDM4 protein and activated the p53 pathway selectively in glioma cells with wild-type p53.

Figure 2.
Figure 2.

CEP-1347 reduces the expression of murine double minute (MDM) 4 and activates the p53 pathway selectively in glioma cells with wild-type p53. The indicated cell lines (A172, U87, T98G) treated without or with CEP-1347 at 500 nM for 3 days were subjected to western blot (A) and quantitative RT-PCR (B) analyses for the expression of MDM4, MDM2, p53, and cyclin dependent kinase inhibitor 1A (CDKN1A, p21Waf1/Cip1).

Reduction in MDM4 expression leads to p53 activation in glioma cells with wild-type p53. Given the well-known capacity of MDM4 to promote MDM2-dependent ubiquitination and degradation of p53 (29), the results so far suggested that CEP-1347 activated the p53 pathway in glioma cells with wild-type p53 by alleviating the negative impact of MDM4 on p53 expression. However, to the best of our knowledge, it remains to be determined whether increased MDM4 expression in glioma cells with wild-type p53 actually plays a pivotal role in the negative regulation of p53. We addressed this question using A172 cells as a model and knocked-down MDM4 overexpressed in A172 cells using independent siRNAs directed against MDM4. The results of the knockdown experiments showed that the expression of p53 and its target genes increased in reverse proportion to the expression level of MDM4 (Figure 3). These results demonstrated that increased MDM4 expression plays an essential role in keeping p53 from being activated in glioma cells with wild-type p53 and that reduction in MDM4 expression is sufficient to activate the p53 pathway in those cells. These findings strongly suggest, though they do not necessarily exclude other mechanisms of p53 activation by CEP-1347, that reduced MDM4 expression mediates CEP-1347-induced p53 activation in glioma cells with wild-type p53.

Figure 3.
Figure 3.

Knockdown of murine double minute (MDM) 4 is sufficient to trigger the activation of the p53 pathway in glioma cells with wild-type p53. A172 glioma cells overexpressing MDM4 with wild-type p53 were transfected either with the indicated siRNA against MDM4 (#1, #2, and #3) or with the control RNA (siCt). After being cultured for 3 and 6 days, the cells were subjected to quantitative RT-PCR (A) and western blot (B) analyses, respectively, for the expression of MDM4, MDM2, p53, and cyclin dependent kinase inhibitor 1A (CDKN1A, p21Waf1/Cip1).

CEP-1347 preferentially inhibits the growth of glioma cells with wild-type p53. The ability of CEP-1347 to activate the p53 pathway by reducing MDM4 expression in wild-type p53 cells demonstrated in this study prompted us to examine whether CEP-1347 selectively inhibits the proliferation and/or survival of cells expressing both wild-type p53 and MDM4 (Figure 4). First, we treated IMR90 normal human fibroblasts with CEP-1347 at concentrations up to 500 nM and confirmed that CEP-1347 did not inhibit their growth at these concentrations, consistent with our previous report (15). We then treated A172, U87, and T98G cells with CEP-1347 within this concentration range. CEP-1347 significantly inhibited the growth and induced death of A172 cells, the highest expressor of MDM4, at as low as 125 nM and that of U87 cells at 250 nM. Unexpectedly, CEP-1347 also inhibited the growth of T98G cells, albeit more mildly than that of U87, despite the inability of CEP-1347 to activate the p53 pathway in T98G cells, indicating that CEP-1347 may also activate a p53-independent growth inhibitory mechanism in glioma cells. Together, the results indicated that CEP-1347 preferentially inhibited the growth of glioma cells with wild-type p53 without showing growth inhibitory effects on normal cells.

Figure 4.
Figure 4.

CEP-1347 preferentially inhibits the growth of glioma cells with wild-type p53. (A) IMR90 normal human fibroblasts and glioma cells (A172, U87, and T98G) treated for 3 days with CEP-1347 at the indicated concentrations were subjected to the dye exclusion assay to determine the numbers of viable and dead cells (upper panels), as well as the percentage of dead cells (lower panels). (B) Cell-growth curves were determined by the dye exclusion assay for the indicated glioma cells treated without (Control) or with 500 nM CEP-1347. The values in the graphs represent mean±standard deviation of three independent experiments.

Discussion

Reactivating p53 activity in human cancers with wild-type p53, which account for nearly half of all human cancers, has been drawing attention as a potential strategy to treat cancer and, in this respect, MDM2 has been by far the most preferred molecular target for decades given its direct and pivotal role in the negative regulation of p53 (1-4, 25). Accordingly, a large number of small-molecule MDM2 inhibitors designed to disrupt the protein-protein interaction between p53 and MDM2 have so far been discovered and developed. However, only a few of them have survived clinical evaluation, and the results of clinical trials for MDM2 inhibitors highlighted the potential challenges associated with MDM2 inhibitors, namely, toxicity to normal tissues (1, 4, 30). To overcome such difficulties inherent in MDM2 inhibitors, recently, efforts have been made to develop inhibitors of MDM4, which interact with both MDM2 and p53 and are thereby capable of inhibiting p53 both in MDM2-dependent and independent manners. So far, largely two types of MDM4 inhibitors have been developed, those targeting (disrupting) MDM4-p53 interaction and those modulating (reducing) MDM4 expression. The anti-tumor activity of such MDM4 inhibitors alone and in combination with MDM2 inhibition has been explored in preclinical studies with promising results and is to be evaluated in clinical trials in the near future (1, 29).

In a recent study, we demonstrated that CEP-1347 exhibited p53-dependent anti-cancer activity in retinoblastoma cells (15). In the same study, we also discovered that CEP-1347 reduced the expression of MDM4 and activated p53 in multiple retinoblastoma cell lines that expressed MDM4 but failed to activate p53 in one that did not express MDM4, which suggested the possibility that CEP-1347 is a novel MDM4 inhibitor capable of activating p53 as such. However, the low transfectability of retinoblastoma cell lines prevented us from conducting MDM4 knockdown experiments to test the possibility and demonstrate that reduction in MDM4 expression was actually sufficient to cause activation of p53 in retinoblastoma cells. Here in this study, not only did we show that CEP-1347 reduced the expression of MDM4 in glioma cells with wild-type p53 but also, through knockdown experiments, we showed that endogenously expressed MDM4 was required to prevent activation of p53 in glioma cells with wild-type p53 and that reduction in MDM4 expression was indeed sufficient to cause activation of p53 and its downstream pathway in those cells. Although these data alone may not exclude other mechanisms for CEP-1347-mediated p53 activation, the results strongly suggested that CEP-1347 activated p53 in glioma cells, at least in part, by inhibiting MDM4 expression.

The present study is also significant in that it demonstrated the inhibitory activity of CEP-1347 on MDM4 expression in cancer cells other than retinoblastoma cells. Since the exact mechanism by which CEP-1347 inhibits MDM4 expression remains poorly understood, we were unable to exclude, with our previous study alone, the possibility that CEP-1347 targeted a mechanism involved in the regulation of MDM4 expression unique to retinoblastoma cells (15). In this scenario, CEP-1347 would have worked as an MDM4 inhibitor exclusively in retinoblastoma cells. However, the results of the present study clearly indicated that CEP-1347 targeted a mechanism of MDM4 expression operative across different types of cancer cells and thereby suggest that CEP-1347 may possibly act as an MDM4 inhibitor in a variety of human cancer cells with wild-type p53.

In terms of therapeutic implications, the results of the present study appear to have important implications for the treatment of glioblastoma, the most malignant form of glioma, for a number of reasons. First, the rate of p53 mutation in glioblastoma is relatively low, with approximately one-fourth of glioblastoma cases having a p53 mutation (31, 32). Second, MDM4 mRNA is reportedly expressed in virtually all glioblastoma cases (23). Although amplification of the MDM4 gene occurs in a rather limited subpopulation (below 10%) of glioblastoma cases (23, 24), recent studies have demonstrated that post-transcriptional regulation of MDM4 expression by miRNAs contributes to increased MDM4 protein expression in colon, lung, and pancreatic cancer cells (25-27), which imply that increased MDM4 protein expression may also occur in glioma cells without MDM4 gene amplification. The functional significance of MDM4 expression in the regulation of p53 expression in glioma cells has not been reported so far, but we demonstrated for the first time that knockdown of MDM4 expression leads to the activation of wild-type p53 in glioma cells. Thus, the currently available data together suggest that glioblastoma is a good candidate in which to test the therapeutic impact of CEP-1347 as an MDM4 inhibitor.

In considering its clinical application for the treatment of human cancers including glioblastoma, the greatest advantage of CEP-1347 is that the safety and pharmacokinetic information of the drug in humans is already available. A previous pharmacokinetic study on human subjects showed that the mean maximal plasma concentration of CEP-1347 reached as high as ~700 nM when the subjects were medicated with 50 mg CEP-1347 twice daily (28), a daily dose which was tolerated for up to 24 months by participants in a preceding clinical trial (7). In accordance with such clinical data, we confirmed in this study that CEP-1347 up to 500 nM did not show growth inhibitory effects on normal human fibroblasts. Yet CEP-1347 did inhibit the growth of glioma cells at concentrations lower than 500 nM, suggesting that there is a therapeutic window. Another strength of CEP-1347 in the treatment of brain tumors is that it penetrates the blood-brain barrier (33). These characteristics of CEP-1347 makes it an attractive drug candidate in the treatment of glioblastoma and thus warrants preclinical evaluation of its therapeutic potential in animal models of glioblastoma.

In conclusion, we have shown for the first time in this study that MDM4 expression is required for the inactivation of p53 in glioma cells with wild-type p53 and that CEP-1347 reduces MDM4 protein expression in glioma cells with wild-type p53, thereby activating the p53 pathway and effectively inhibiting their growth. Our results suggest that the use of CEP-1347 as an MDM4 inhibitor may possibly be a viable approach to activating p53 in human cancers with wild-type p53, including retinoblastoma and glioblastoma.

Acknowledgements

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Footnotes

  • Authors’ Contributions

    MO and CK designed the research. YM and MO performed the experiments under the supervision of MO and CK. YM, YN-S, KT, SS, AS, K-IM, YS, CK, and MO were involved in data interpretation. YM, MO, and CK drafted the manuscript and prepared the figures. YN-S, KT, SS, AS, K-IM, and YS reviewed the draft and based on their inputs, YM, MO, and CK edited the manuscript. All Authors read and approved the final version of the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received August 1, 2022.
  • Revision received September 1, 2022.
  • Accepted September 5, 2022.

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CEP-1347 Targets MDM4 Protein Expression to Activate p53 and Inhibit the Growth of Glioma Cells
YUTA MITOBE, YURIKA NAKAGAWA-SAITO, KEITA TOGASHI, SHUHEI SUZUKI, ASUKA SUGAI, KEN-ICHIRO MATSUDA, YUKIHIKO SONODA, CHIFUMI KITANAKA, MASASHI OKADA
Anticancer Research Oct 2022, 42 (10) 4727-4733; DOI: 10.21873/anticanres.15977
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