Inhibition of cyclin E1 overcomes temozolomide resistance in glioblastoma by Mcl‐1 degradation

Huaxin Liang | Zhuo Chen | Libo Sun


As the most common and most lethal primary brain tumor, glioblastoma (GBM) leads to 12 to 14 000 deaths each year in the US alone.1 Current therapy for GBM is usually constrained in surgical resection, radiation, and temozolomide chemotherapy, and the median survival following treatment is very poor, approximately 12 to 15 months.2 Temozolomide (TMZ) is an oral alkylating agent used to treat GBM. However, at least 50% of TMZ treated patients have poor drug response, which is termed as drug resistance.2 TMZ resistance leads to adverse reactions, longer chemotherapy cycle, and affects the survival and the quality of life of patients with GBM. Therefore, the TMZ resistance is a major obstacle toward the achievement of better survival in the treatment of this disease. Understanding the underlying molecular mechanism of the TMZ resistance is very important to improve the clinical benefit and the survival of patients with GBM.
As a DNA alkylating agent, TMZ is known to induce cell‐cycle arrest at G2/M and to eventually lead to apoptosis.3 The failure of many of chemotherapeutic agents reflects, on a cellular level, an inability of these drugs to induce apoptosis.4,5 The cell‐cycle and apoptosis are intimately related, as evidenced by the central role of p53, both in cell‐cycle arrest and in the induction of apoptosis.4 Approximately 30% of neuroblastomas exhibit aberrations in genes which regulate the G1 checkpoint.6 Cyclin‐dependent kinases play crucial roles in regulating the stability of cell‐cycle‐related proteins during cell‐cycle progression.7 Numerous studies have identified genetic aberrations in neuroblastoma and malignant rhabdoid tumor that increase cyclin‐dependent kinase (CDK) 4/6 activity.8 Genomic amplification of CCND1 (cyclin D1) and CDK4 is correlated with poor prognosis.8 Overexpression of cyclin E1 is also involved in various types of cancers, including breast, colon, bladder, skin, and lung cancer.9 It was reported that patients with brain cancer have high DNA copy‐number amplification of cyclin E1.10 Besides, cyclin E1 overexpression is a mechanism of drug resistance in multiple cancers, including breast cancer,11 ovarian cancer,12 and hepatocellular carcinoma.13 However, it is still unclear whether dysregulation of cyclin family proteins is involved in the TMZ resistance in GBM.
In this study, we found that the abnormal expression of cylcin E1 (CCNE1) is correlated to the TMZ resistance in GBM. Depletion of cyclin E1 or inhibition of CDK2 can overcome the TMZ resistance invitro and in vivo. As to the mechanism, we found that the expression of cyclin E1 promoted the stabilization of Mcl‐1, and therefore resistant to TMZ‐induced apoptosis. Generally, our results suggested that cyclin E1 inhibition combined with the TMZ might be a potential strategy for GBM therapy.


2.1 | Cell culture and reagents

The GBM cell lines, such as temozolomide (TMZ) sensitive cells line (U‐87, A172), and TMZ resistant cell lines (U‐138MG, LN‐229, and U‐118MG)14 were procured from American Type Culture Collection (Manassas, VA). The cell lines were preserved at 37°C with 5% CO2. Dulbecco’s modified Eagle’s medium (DMEM) consisting of Ham’s F12 medium (1:1; Invitrogen, Shanghai, China) was mixed with 10% fetal bovine serum (FBS; HyClone, Logan, UT). Chemicals, including TMZ, flavopiridol (FLA), UMI‐77, MG132, and dinaciclib (DIN), were purchased from Sigma‐Aldrich (St. Louis, MO). The Mcl‐1 inhibitor S63845 was purchased from APExBIO (Houston, TX).

2.2 | Establishment of TMZ‐resistant GBM cell line

The A172 and U87 TMZ resistant cell line was developed by using the TMZ concentration gradient progressive methods as previous described.15 Briefly, A172 or U87 cells at a concentration of 1× 105/mL were inoculated in TMZ‐free culture medium for 24 hours until they got in the logarithmic phase. Then the culture medium was replaced with that containing low concentration (5 µM) TMZ for 48 hours. Then the culture medium containing drugs and dead cells was discarded. Cells were collected and re‐inoculated in TMZ‐free culture medium to get recovered before the next TMZ treatment. After cells got adjusted to present the TMZ treatment, the concentration of TMZ was increased in turn until cells survived well and developed resistance to the 10 µM TMZ treatment.

2.3 | Transfection of siRNA and plasmid

Transfection of small interfering RNA (siRNA) and plasmid was carried out using Lipofectamine® 2000 (Thermo Fisher, Shanghai, China).16 Plasmid expressing the gene cyclin E1 was produced via insertion of cDNA into pcDNA3.1‐HA vector (Addgene, Cambridge, MA). Mcl‐1 expression plasmid, pcDNA3.1‐Mcl‐1, was purchased from Addgene (#25375). Preliminarily prepared siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) was utilized to knock down the genes cylcin E1 and Mcl‐1.

2.4 | Western blot analysis

Approximately 40 μg of cell line or patient tumor lysate was prepared as previously described,17 separated by electrophoresis on 4% to 12% polyacrylamide gels (Invitrogen), transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Shanghai, China), and probed with primary antibodies: cleaved‐caspase‐3 (#9661), cyclin D1 (#2922), cyclin E1 (#4129), Mcl‐1 (#94296), ubiquitin (#3933; Cell Signaling, Shanghai, China), actin (A2228, Sigma‐Aldrich, Shanghai, China), CDK4 (SC‐70832), CDK6 (SC‐7961), and tubulin (SC‐73242; Santa Cruz, Shanghai, China).

2.5 | Cell‐cycle analysis

Cell lines were plated in duplicate in 35 mm dishes and treated 24 hours later with the indicated concentrations of TMZ or with a dimethyl sulfoxide (DMSO) control. At 24 hours posttreatment, cells were gently harvested and fixed overnight in 70% ethanol. Cells were then washed in phosphate‐buffered saline (PBS), stained with 1 µg/µL propidium iodide (PI, Thermo Fisher), and assayed for DNA content on an Attune Acoustic Focusing Cytometer (Invitrogen). Analysis was carried out using VenturiOne software (Applied Cytometry, Dinnington, UK).

2.6 | Cell death analysis

The apoptosis of GBM cells was analyzed by Hoechst 33258 staining (3.7% formaldehyde, 0.5% Nonidet P‐40, and 10 µg/mL Hoechst 33258 (Invitrogen)), followed microscopic visualization of condensed chromatin and micronucleation as described.17
The cell viability was determined by MTT (3‐[4, 5‐dimethylthia- zol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide) assay (Promega) as described by manufacturer. Briefly, cells were seeded into 96‐well plates (BD Biosciences, Bedford, MA) at a cell density of 1 × 103 cells/ well in DMEM. Cell growth was assayed for five consecutive days by addition of 20 μL of MTT (5 mg/mL; Sigma‐Aldrich) to each well and the plate was incubated at 37°C for 4 hours. The proliferation assays were performed for four consecutive days. Later, the reaction was stopped by addition of 200 μL DMSO (Sigma‐Aldrich). Optical density (OD) was measured at 570 nm with microplate reader 680 (Bio‐Rad, Hercules, CA).

2.7 | Xenograft therapeutic trials

The animal studies were approved by the Institutional Animal Ethics Committee (IEC) of Jilin University and experiments were performed in accordance with the Animal Ethics guidelines of Jilin University. For invivo tumorigenicity assays, severely immuno‐compromised female NOD‐SCID mice were used. Briefly, 1 × 106 of parental A172 parental and TMZ‐resistant cells were injected subcutaneously in NOD‐SCID mice (n = 5). We began monitoring the tumor development and mice weight every other day mice at 7 days after cell injection, when tumor size reached 50 mm3, and counted it as day 1 for the further schedule of the drug treatment. The mice were received intraperitoneal injection of TMZ (25 mg/kg), FLA (3 mg/kg), TMZ + FLA (25 mg/kg, and 3 mg/kg, respectively), or PBS as a negative control (control). The drug injection was administrated three times a week for total of 2 weeks. The tumor volume was determined using the formula: 4/3π (√major axis/2 × minor axis/2). Mice were killed at day 17, and tumors were dissected for Western blot or fixed in 10% formalin and embedded in paraffin for terminal‐deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining.

2.8 | Statistical analysis

Statistical analyses of t‐test or one‐way analysis of variance (ANOVA) were performed using GraphPad Prism V software (San Diego, CA). Every experiment was conducted for at least three times. P values < 0.05 were considered to be statistically significant. Means + SD were displayed in the figures. 3 | RESULTS 3.1 | TMZ resistant GBM cells have less apoptosis and cell‐cycle arrest To study the mechanism of TMZ resistance in GBM, we generated the TMZ resistant GBM cell lines by treating cells with five consecutive rounds of TMZ, including A172, and U87. The resistant cells transfected with control or cyclin E1 siRNA, cells (‐R cells) has less cell loss than the parental cells (‐P cells), when treated with the same doses of TMZ (Figure 1A). As TMZ is a chemotherapy drug, and induced GBM cell death by apoptosis,2 we tested 20 µM TMZ‐induced apoptosis in A173 and U87 parental and resistant cells using Hochest 33258 nuclear staining. We found that TMZ significantly induced apoptosis in A172P and U87P cells, which was reduced in A172R and U87R cells (Figure 1B,C). The parental and resistant cell did not show any difference in cell proliferation tested by the MTT assay (Figure 1D). Furthermore, TMZ‐induced cleavage of caspase‐3 in A172P and U87P cells, which is absent in resistant cells (Figure 1E), further confirmed that apoptotic signals were compromised in the TMZ resistant cells. To further investigate the difference between parental and resistant cells, we analyzed the cell‐cycle changes in response to the TMZ treatment. Consistently, TMZ treatment in A172 and U87 parental cells have higher percentage of sub G0 phase when compared with resistant cells (Figure 1F), further, confirming that TMZ‐induced apoptosis was reduced in the TMZ resistant cells. Interestingly, the TMZ parental cells showed significantly cell‐cycle changes in response to the TMZ treatment (Figure 1F), which is not observed in TMZ resistant cells (Figure 1F), indicating that the dysfunction of cell‐cycle might contribute the TMZ resistance in GBM cells. 3.2 | Expression of cyclin E1 contributes to TMZ resistance in GBM As cyclin family proteins are vital in modulating cell‐cycle, we tested their expression in resistant cells and parental cells. As showed, the expression of cyclin E1 was higher in resistant cells than parental cells, but not cyclin D1, CDK4, or CDK6 (Figure 2A). Multiple GBM cell lines are known to contain TMZ resistant cells and several acquired TMZ resistant GBM cell lines have been developed for use in experiments designed to define the mechan- ism of TMZ resistance and the testing of potential therapeutics. We therefore analyzed the expression of cyclin family proteins in multiple TMZ resistant and sensitive cell lines, including A172, U87, U138MG, LN‐229, and U118MG. Consistently, the expression of cyclin E1 in TMZ resistant cell lines was also higher than that in TMZ sensitive cell lines (Figure 2B), suggesting that upregulation of cyclin E1 might be the cause of TMZ resistance. To confirm our hypothesis, we depleted cyclin E1 by siRNA transfection, and found that knockdown of cyclin E1 in A172R cells re‐sensitized the cells to the TMZ treatment (Figure 2C). The TMZ treatment also induced higher apoptosis in A172R cells transfected with cyclin E1 siRNA (Figure 2D,E). Reversely, overexpression of cyclin E1 in A172P cells phenocopied the A172R cells, which showed resistant to TMZ‐induced cell loss and apoptosis (Figure 2F‐H). These results indicate that GBM cells gain resistance to TMZ by upregulation of cyclin E1. 3.3 | Inhibition of cyclin E1 sensitized TMZ resistant GBM We next assessed if Cyclin E1 inhibition could resensitize the resistant cells to the TMZ treatment. CDK2 is an important coordinator of cyclin E1 in cell‐cycle control,18 we therefore tested the cyclin E1 inhibition by using a pan‐CDK inhibitor, flavopiridol (FLA). A172R cells were treated with control DMSO, FLA, TMZ, or combination therapy for 24 hours and apoptosis was measured by the Hochest staining. When compared with control cells, we found a modest increase in apoptosis in A172R cells (Figure 3A) following individual FLA or TMZ treatment, with the highest level of apoptosis observed in response to treatment with combination therapy (Figure 3A). Furthermore, the combination treatment also signifi- cantly induced the highest level of caspase‐3 (3B). To further confirm the effect of cyclin E1 inhibition, we also used another CDK2 inhibitor, dinaciclib (DIN).19 Consistently, we found the combination with DIN also sensitized the TMZ resistant A172 cells to TZM induced cell loss and apoptosis (Figure 3C,D). These data further support the Cyclin E1 modulated the TMZ resistance in GBM cells. 3.4 | Cyclin E1 modulated the expression of Mcl‐1 in TMZ resistant cells We next examined the mechanism of cyclin E1 induction in the resistant cells. It was reported that cyclin E1 inhibition overcomes the drug resistance in multiple cancer by suppression of Mcl‐1 expression,13,20 w therefore investigated the expression of Mcl‐1 in TMZ parental and resistant cells. We found that the expression of Mcl‐1 in A172P cells was downregulated, but did not change in A172R cells when treated with the same dosage of TMZ (Figure 4A). Similarly, the downregulation of Mcl‐1 in A172P cells was also suppressed by cyclin E1 overexpression (Figure 4B). Furthermore, the expression of Mcl‐1 in A172R cells was suppressed by the supplement of cyclin E1 inhibitor (Figure 4C), indicating that cyclin E1 regulated the Mcl‐1 expression in TMZ resistant cells. However, the TMZ treatment did not significantly change the mRNA level of Mcl‐1 in A173 parental or resistant cells (Figure 4D), suggesting that cyclin E1 did not change the mRNA level of Mcl‐1. We therefore tested the expression of Mcl‐1 in posttranscriptional level. Supplement of protease inhibitor, MG132, suppressed the down-regulation of Mcl‐1 in A172P cells treated with TMZ, or in A172R cells treated with cyclin E1 inhibitor (Figure 4E,F), indicating that cyclin E1 modulated the Mcl‐1 by inhibiting its degradation. As degradation of a protein is via the ubiquitin‐proteasome pathway, we, therefore, studied the ubiquitination of Mcl‐1 in A172 cells upon the TMZ treatment. We found that TMZ treatment increased the ubiquitination of Mcl‐1, which was suppressed by overexpression of cyclin E1 (Figure 4G). Collectively, our results suggested that cyclin E1 modulated the Mcl‐1 expression by inhibiting its posttranscriptional degradation. 3.5 | Mcl‐1 mediated the TMZ resistance in GBM induced by cyclin E1 To further confirm the role of Mcl‐1 in cyclin E1 mediated TMZ resistance, we depleted the Mcl‐1 in A172R cells by siRNA transfection. We found that depletion of Mcl‐1 sensitized the A172 resistant cells to TMZ‐induced apoptosis (Figure 4A,B). Reversely, enhanced expression of Mcl‐1 in A172R cells compromised the apoptosis induced by a combination of TMZ and FLA (Figure 4C,D). To further confirm the function of Mcl‐1 in TMZ resistance, we used two different latest Mcl‐1 inhibitor to treat TMZ resistant A172 cells, including S63745 and UMI‐ 77.21,22 Our results showed Mcl‐1 inhibitors can induce higher apoptosis than TMZ in A172R cells (Figure 4E), and combination of Mcl‐1 inhibitors with TMZ achieved maximal killing effects (Figure 4E). Together these results indicate that abnormal expression of Mcl‐1 is essential for cyclin E1 mediated TMZ resistance in GBM cells (Figure 5). 3.6 | Inhibition of cyclin E1 enhanced the killing effect of TMZ in vivo A172 parental and resistant cells subcutaneously into the flanks of the NOD‐SCID mice. After 7 days, the mice were randomly divided into six groups and administered an intraperitoneal injection of TMZ, FLA, TMZ + FLA, or PBS as a negative control (control). After the drug treatment, the tumor size significantly shrank in the parental tumor after the TMZ injection (Figure 6A). However, the tumor grown from resistant cells did not showed a significant response to the TMZ treatment (Figure 6A). And the combination of TMZ with FLA treatment dramatically suppressed the tumor growth, compared with the TMZ or FLA single treatment in resistant tumors (Figure 6A). We also monitored the body weight change of mice, and found that either single treatment or combination treatment did not significantly change the body weight of mice (Figure 6B), suggesting the safety of FLA and TMZ combination. We also analyzed the caspase‐3 and Mcl‐1 expression in different tumors after treatment, and found that the TMZ treatment actually suppressed the Mcl‐1 expression and induced cleavage of caspase‐3 in A172 parental tumors, which was abrogated in A172 resistant tumors (Figure 6C). And combination of TMZ with FLA rescued the caspase‐3 activation in A172 resistant tumor, and also suppressed the Mcl‐1 expression (Figure 6D). Finally, the TUNEL staining results indicated that the combination treatment can reactivate the apoptosis in A172 resistant tumors (Figure 6E). Therefore, inhibition of cyclin E1 could resensitize the GBM resistant cells to TMZ treatment in vivo. 4 | DISCUSSION Drug resistance is usually an obstacle for the most cancer therapy, including TMZ therapy in GBM. In our study, we reported that the abnormal expression of cyclin E1 led to TMZ resistance in GBM cells. Inhibition the pan‐CDK by its inhibitor, flavopiridol, can significantly improve the efficacy of TMZ both in vitro and in vivo. Since the antitumor effect of TMZ is schedule‐dependent with multiple administrations being more effective than a single treatment, our findings provide a new rationale of design for combination therapy for GBM. With increased understanding of the impact chemotherapeutic agents on the cell‐cycle, it is well recognized that the dysfunction of cell‐cycle is one of the major causes of drug resistance in cancer therapy.4,11 Cyclin D1 and cyclin E1, as vital regulatory factors of the G1‐S phase cell‐cycle progression, are frequently constitutive expressed and associated with pathogenesis and tumorigenesis in most human cancers and they have been regarded as promising targets for cancer therapy.4,8,12 However, we found that the only the expression of cyclin E1 shows different in TMZ sensitive and resistant cells. Inhibition or depletion cyclin E1 re‐sensitized the TMZ resistant GBM cells to TMZ treatment, suggesting that individual cell‐cycle regulators may still play specific functional roles. Cyclin E1 has been reported to be associated with chemoresistance. In ovarian carcinomas, high cyclin E1 copy number and protein expression were associated with primary chemoresistance.23 More- over, overexpression of cyclin E1 reduced the sensitivity to seliciclib in myeloma,24 and is associated with cisplatin resistance in bladder cancer.25 There is a significant association between cyclin E overexpression and the prognosis of lung cancer. It is believed the increased expression of cyclin E correlated with poorer prognosis.26 Our result showed that cyclin E1 is upregulated in TMZ‐resistant glioma cells and its knockdown suppressed cell viability and reduced the resistance to TMZ. Thus, the suppression of cyclin E1 may serve as a pharmacodynamic marker of TMZ efficacy in GBM, and further study on cyclin E1 expression in GBM tumor tissues prospectively collected from patients would help validate our findings. Since the dysregulated cell‐cycle control in cancer cells has been extensively studied, it was reported that most clinical trials on single‐ agent treatment with CDK inhibitors or other cell‐cycle regulators have significant antitumor efficacy. Recent developments of CDK inhibitors for cancer treatment have focused on selective CDK4/6 inhibitors, such as palbociclib, abemaciclib, and ribociclib.27,28 However, little is known about other CDK inhibitor on the GBM therapy. Our data also found that use of CDK2 inhibitors can overcome the TMZ resistance in GBM. Pan‐CDK inhibitors such as flavopiridol or dinaciclib may have better antitumor activity through both cell‐cycle regulation and the suppression of the expression of antiapoptotic proteins, including Mcl‐1, Bcl‐XL, and XIAP.20,29 It was also reported that flavopiridol combination with TMZ showed enhanced cytotoxicity in a glioblastoma cell line and sensitized xenografted mice to TMZ.30 A potential concern about using pan‐CDK inhibitors is the off‐target adverse events. However, our data indicate that flavopiridol can enhance the antitumor efficacy of TMZ, but without affecting the body weight loss of mice. Future clinical trials should clarify whether specific or multitargeted CDK inhibitors can yield a more favorable therapeutic index in clinics. Mcl‐1, a Bcl‐2 (B‐cell lymphoma‐leukemia 2) homolog, is known to function as an antiapoptotic protein. Results from our study and other investigators support Mcl‐1 as a key mediator of cell survival and drug resistance in GBM.3,31 Tagscherer et al showed that Bcl‐2, Bl‐xL and Bcl‐w inhibitor, ABT‐737, selectively killed the Mcl‐1 low expressed GBM cells.32 Similar observations were made by van Delft et al, showing that Mcl‐1 high expression led to apoptosis defect in GBM cells.33 In our study, we found the Mcl‐1 expression in TMZ resistant cells can not be suppressed by the TMZ treatment, which rendered the resistance phenotype. Cyclin E1 was reported to phosphorylated the PEST domain of MCl‐1, and protected it from ubiquitin‐proteasome degrada- tion.34 Consistently, we also found cyclin E1 expression sup- pressed the Mcl‐1 ubiquitination, and stabilized it in the TMZ resistant cells. Inhibition of cyclin E1 by flavopiridol lead to degradation of Mcl‐1, and sensitized the GBM cell to TMZ‐ induced apoptosis. These findings are consistent with the data presented before, which showed that flavopiridol induces cancer cell apoptosis through downregulation of Mcl‐1.20,35 Therefore, Mcl‐1 expression can serve as an alternative type of combination therapy to overcome the TMZ resistance in GBM. In conclusion, this study indicates that flavopiridol may be an effective drug in the treatment of GBM. GBM cells are known to use multiple survival pathways to avoid TMZ‐induced apoptosis, which renders them resistant to a variety of therapeutic interventions. However, it appears that inhibition of cyclin E1 or Mcl‐1 may overcome one or more of these protective mechanisms, making them as attractive target for the development of new therapeutics. The data in this report are in agreement with this hypothesis and support clinical trials of flavopiridol and Mcl‐1 inhibitors for treatment of GBM ( REFERENCES 1. Hess KR, Broglio KR, Bondy ML. Adult glioma incidence trends in the United States, 1977‐2000. 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