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E2F8 confers cisplatin resistance to ER+ breast cancer cells via transcriptionally activating MASTL
Biomedicine & Pharmacotherapy 92 (2017) 919–926
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Original article
E2F8 confers cisplatin resistance to ER+ breast cancer cells via transcriptionally activating MASTL Jianjun Tiana,1, Yuting Linb,1, Jianhua Yuc,* a
Clinical Laboratory, Qilu Children’s Hospital of Shandong University, Jinan, 250000, Shandong, China Clinical Laboratory, Beihai Hospital of Yantai, Yantai, 264000, Shandong, China c Clinical Laboratory, The 148th Hospital of Chinese People’s Liberation Army, Zibo, 255000, Shandong, China b
A R T I C L E I N F O
Article history: Received 12 April 2017 Received in revised form 15 May 2017 Accepted 24 May 2017 Keywords: E2F8 Cisplatin resistance Breast cancer MASTL Cell cycle
A B S T R A C T
MASTL (microtubule-associated serine/threonine kinase-like) is a critical kinase modulating mitotic entry. In this study, we investigated the mechanism of its dysregulation in breast cancer and its involvement in cisplatin resistance in ER+ breast cancer cells. Data mining in Kaplan-Meier Plotter showed that high MASTL expression was associated with worse distant metastasis free survival (DMFS) and relapse free survival (RFS) in ER+ breast cancer patients. In TCGA breast cancer cohort (TCGA-BRCA), MASTL was strongly co-upregulated with E2F8. High E2F8 expression was also strongly associated with unfavorable DMFS and RFS in ER+ breast cancer patients. Promoter scanning in JASPAR Database showed that the MASTL promoter region has a highly possible E2F8 binding site upstream the TSS site. The following western blot, dual luciferase assay and ChIP-qPCR validated this binding site. In MCF-7 cells, E2F8 overexpression alleviated cisplatin induced cell apoptosis by shortening G2/M arrest and promoting mitotic entry, the effect of which was largely canceled by inhibiting MASTL. Therefore, we infer that E2F8 can shorten cisplatin induced G2/M arrest by promoting MASTL mediated mitotic progression in ER+ breast cancer cells. These findings might help to explain why high MASTL or high E2F8 expression is associated with worse RFS in ER+ breast cancer. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Breast cancer is the leading cancer in women across the world [1]. For the patients with advanced breast cancer, chemotherapy is still the major therapeutic strategy [2,3]. Cisplatin, together with other platinum-based drugs, are most successful and commonly used chemotherapeutic drugs [4]. However, the tumors initially responsive to chemotherapeutic agents usually develop acquired drug resistance after several rounds of therapy, which is the major cause of relapse, metastasis and finally cancer related death [5,6]. MASTL (microtubule-associated serine/threonine kinase-like) is a critical kinase modulating mitotic entry. In brief, MASTL can be activated directly by cyclin B-Cdk1 [7]. Once activated, MASTL supports further phosphorylation of cyclin B-Cdk1 substrates by restraining PP2A [7]. This feedback loop directly boosts CDK1 activity above the threshold required for mitotic entry [8]. One recent study reported that MASTL also acts as a regulator of the
* Corresponding author. E-mail address: [email protected] (J. Yu). Jianjun Tian and Yuting Lin contributed equally to this study.
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http://dx.doi.org/10.1016/j.biopha.2017.05.118 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
DNA damage response (DDR), a cellular surveillance mechanism [9]. In fact, the chemotherapeutic drugs are usually DNA damaging agents that activate DDR and following DNA repair, cell cycle checkpoint, and cell death [10]. Therefore, it is generally recognized that the dysregulated genes related to DDR are critically involved in cancer progression and therapy. In breast cancer, MASTL overexpression induces oncogenic properties such as transformation and invasiveness of the cancer cells [8]. In breast cancer, MASTL upregulation is correlated with a more advanced clinical stage [11]. However, the mechanism underlying its dysregulation in breast cancer is not clear. The E2Fs are a family of transcription factors that can bind to target promoters and regulate their transcription [12]. Traditionally, E2F1-E2F3 are considered as transcription activators, while E2F4E2F8 act as repressors [13]. However, in breast cancer, one recent study found that E2F8 acts as a transcription activator that upregulates the expression of CCNE1 and CCNE2 via directly interacting with their respective gene promoter, thereby promoting cancer cell proliferation [14]. Another recent study reported that in prostate cancer, knockdown of E2F8 was sufficient to suppress cell growth by inducing G2/M arrest [15]. These findings suggest that E2F8 might be an important gene modulating cell cycle progression.
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In this study, we found that E2F8 can transcriptionally activate MASTL via binding to its promoter and this regulative axis shortens cisplatin induced G2/M arrest in ER+ breast cancer cells. 2. Materials and methods 2.1. Microarray reanalysis The raw microarray data (GDS5666) [16] that compared gene expression profiles among the parental mammary tumor cell line 4T1 and 4T1 derived metastatic populations isolated from liver, lung or bone were downloaded from GEO datasets. The raw data of bone-aggressive explant and primary tumor explant were reanalyzed to identify the dysregulated genes. 2.2. Bioinformatic data mining To pool available annotated genomic data that assessed the association between MASTL/E2F8 expression and distant metastasis free survival (DMFS) and relapse-free survival (RFS) in breast cancer patients, data mining was performed using Kaplan-Meier Plotter (http://kmplot.com/analysis/), an online database that provides assessment of the effect of 54,675 genes on survival using 10,293 cancer samples, including 22,277 genes in 5143 breast cancer samples [17,18]. The survival curve were generated by using the JetSet best probe set with automatically selected best cutoff. The genes co-expressed with MASTL in TCGA breast cancer cohort (TCGA-BRCA) were identified using cBioPortal for Cancer
Genomics (http://cbioportal.org) [19]. The heat map of MASTL and E2F8, and their correlation were further studied using UCSC Xena Browser (http://xena.ucsc.edu/). The promoter sequence of MASTL was obtained from GeneCopoeia (> HPRM37364, NM_001172303) and was given in Supplementary material 1. The possible E2F8 binding sites in MASTL promoter region were predicted by using the JASPAR Database (http://jaspar.genereg.net/). The TSS site and the predicted E2F8 binding sites were marked in red font in Supplementary material 1. 2.3. Cell culture and transfection The human ER+ breast cancer cell line MCF-7 and BT474 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 10 units/mL penicillin, and 10 mg/mL streptomycin. MASTL Human Lentifect purified lentiviral particles (NM_001172303.1) and E2F8 Human ORFeome purified lentiviral particles, and lentiviral MASTL shRNA (HSH067101-LVRH1GP, based on psi-LVRH1GP vector) were obtained from Genecopoeia. Cells were infected with the lentiviral particles in the presence of 5 mg/mL polybrene. 2.4. Western blot analysis Conventional western blotting was performed as described previously [20]. Antibodies against E2F8, phosphor-histone
Fig. 1. High MASTL expression is associated with worse DMFS and RFS in ER+ breast cancer. A. The heap map the 30 most upregulated genes in 4T1 derived bone-aggressive explants compared to primary tumor. Red: up-regulation; Blue: down-regulation. Raw microarray data was obtained from GEO dataset (accession: GDS5666). B–E. Kaplan-Meier plots of the association between MASTL expression and DMFS (B–C) and RFS (D-E) in ER+ (B and D) or ER- (C and E) breast cancer patients. Data was obtained by using Kaplan-Meier Plotter.
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H3Ser10, MASTL, CCNE1, CCND2, CCND1 and b-actin were obtained from Abcam (Cambridge, MA, USA). The protein bands were visualized by using the SuperSignalTM West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, lL, USA).
system with a luminometer (Promega, Madison, WI, USA) following the manufacturer’s instructions.
2.5. QRT-PCR
ChIPassaywasperformedbyusingtheUpstate-ChIPAssayKit(Lake Placid, NY, USA) following the manufacturer's instructions. Equal aliquots of chromatin supernatants from MCF-7 cells were subjected to immunoprecipitation with 1 mg of normal goat IgG or anti-E2F8 (ab109596,Abcam)overnightat4 Cwithrotation.Afterreversecrosslink of protein/DNA complexes to free DNA, the ChIP-enriched DNA was analyzed by qRT-PCR using the ABI 7900HT sequence detection system and SYBR green master mix. Primers used were: GAPDH, 50 agcgcaggcctcaagacctt-'3 (forward) and 50 -aagaagatgcggctgactgt-30 (reverse), and MASTL promoter, 50 -gacggagtttcgttctttcg-30 (forward) and 50 -ggcccagtcagaagatttca-30 (reverse).
24 h after infection of lentiviral E2F8 expression particles or the empty control, MCF-7 and BT474 cells were lysed for exaction of total RNA, with the using of Trizol Reagent (Invitrogen). Then, cDNA was obtained by reverse transcription using a First-Strand Synthesis Kit (Invitrogen). To detect E2F8 mRNA expression, qRTPCR analysis was performed on an ABI 7900HT sequence detector using gene specific primers (E2F8, forward: 50 -CCAACCCTGCTGTGAATA-30 ; reverse: 50 -TTTCTGGCTCATTACCCT-30 ) and TaqMan Universal PCR Master Mix according to the directions of manufacturer (Applied Biosystems, Foster City, CA, USA). The 2DDCt method was used to calculate the relative expression. 2.6. Dual luciferase assay MASTL promoter fragments (1309 to +173, 1000 to +173, 700 to +173, 400 to +173, 300 to +173 and 100 to +173) were PCR amplified from the MASTL promoter clone (HPRM37364) and then were inserted into the XhoI-Hind III site of the pGL3-basic luciferase reporter vector respectively. Then, dual luciferase assay was performed as described previously [20]. In brief, HEK-293 cells were seeded into 12-well plates at a density of 2 105 cells per well and were further infected with lentiviral E2F8 expression particles or the empty control. 24 h later, the cells were co-transfected with 1.5 mg luciferase construct plasmid or the empty reporter vector DNA and 0.05 mg phRL-TK by using Superfectin (Qiagen, Valencia, CA, USA). 24 h later, the cells were lyzed and luciferase activity of the lysate was measured using the dual-luciferase reporter assay
2.7. Chromatin immunoprecipitation assay (ChIP)
2.8. Flow cytometric analysis of active caspase-9 24 h after infection, MCF-7 cells were subjected to 5 mM cisplatin treatment for another 48 h. After the treatment, the proportion of cells with activated caspase-9 was determined using the GaspGLOW TM Fluorescein Active Caspase-9 Staining Kit (Biovision, Mountain View, CA, USA) according to the manufacturer’s protocol. Fluorescence was excited with the 488-nm line of an argon laser on an EPICS XL flowcytometer (Beckman Coulter, Fullerton, CA, USA). 2.9. Cell cycle analysis MCF-7 after indicating treatment and at indicating time points were collected, washed and fixed with ice-cold methanol. Then the cells were stained with the Cellometer PI Cell Cycle Kit (Nexcelom Bioscience, Lawrence, MA, USA) according to the protocol
Fig. 2. MASTL and E2F8 are co-upregulated in breast cancer. A. The top genes positively correlated to MASTL expression in TCGA-BRCA. B. The correlation between MASTL and E2F8 in TCGA-BRCA. Analysis was performed by using cBioPortal. C–F. The heat map (C–D) and regression analysis (E–F) of the expression between MASTL and E2F8 in ER+ and ER- subgroups in TCGA-BRCA. Analysis was performed using UCSC Xena browser.
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recommended by the manufacturer. After that, the cells were loaded into the cellometer counting chamber and the cell cycle distribution was determined using Partec flow cytometer.
3. Results 3.1. High MASTL expression is associated with worse DMFS and RFS in ER+ breast cancer
2.10. CCK-8 assay of cell viability 24 h after indicating transfection, MCF-7 cells were seeded in a 96-well plate and were further treated with varying concentrations of cisplatin for 48 h. After that, cell viability was measured using the Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to manufacturer’s instruction. In brief, CCK-8 solution was added to each well and the plate was placed in a cell incubator for 4 h. Cell viability was reflected by the absorbance at 450 nm determined by a 96-well spectrophotometry. 2.11. Statistical analysis Data were presented in the form of means standard deviation (SD) with data from three repeats. Data were analyzed for statistical significance by two-tailed Student’s t-test or ANOVA with Student-Newman-Keuls test as a post hoc test. p value of <0.05 was considered statistically significant.
One recent study reported that MASTL has oncogenic properties in breast cancer and its upregulation is associated with cancer progression [11]. By reanalysis of the raw data of one previous microarray (GDS5666), we observed that MASTL upregulation is significantly associated with bone metastasis compared to primary tumor (Fig. 1A, red arrow). Then, we explored whether there is any association between MASTL expression and survival outcomes in breast cancer patients by data mining in Kaplan-Meier Plotter. The results showed that high MASTL expression was associated with worse DMFS and RFS in ER+ breast cancer patients (Fig. 1B and D), but not in ER- patients (Fig. 1C and E). 3.2. MASTL and E2F8 are co-upregulated in breast cancer Then, we tried to explore the underlying mechanism of MASTL dysregulation in breast cancer. By data mining in TCGA-BRCA using the cBioPortal, we found that E2F8 was positively correlated with
Fig. 3. E2F8 transcriptionally activates MASTL via binding to its promoter. A. Western blot analysis of E2F8 expression in MCF-7 and BT474 cells 24 h after infection of lentiviral E2F8 expression particles or the empty control. B-C. QRT-PCR analysis of MASTL mRNA (B) and western blotting of MASTL protein (C) expression in MCF-7 and BT474 cells 36 h after infection of lentiviral E2F8 expression particles or the empty control. D. Predicted E2F8 binding site in MASTL promoter. E. The luciferase reporter constructs carrying truncated MASTL promoter sequences were introduced into HEK-293 cells pre-infected with lentiviral E2F8 expression particles or the empty control. Luciferase activity was measured 24 h post-transfection. F. Fold-enrichment of E2F8 binding at MASTL promoter relative to background in MCF-7 cells was measured by CHIP-qPCR. Upon normalization to GAPDH, results were expressed as n-fold compared to IgG. G. The heat map of E2F8, CCNE1, CCNE2 and CCND1 expression in ER+ subgroup in TCGA-BRCA. H. Summary of regression analysis of the correlation between E2F8 and CCNE1, between E2F8 and CCNE2 and between E2F8 and CCND1. I. Western blotting of E2F8, CCNE1, CCNE2 and CCND1 expression in MCF-7 and BT474 cells 36 h after infection of lentiviral E2F8 expression particles or the empty control. **, p < 0.01.
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MASTL expression (Pearson’ r = 0.66, Fig. 2A–B). To further verify the co-expression in ER+ and ER- patients, their expression profiles in TCGA-BRCA were also checked using the UCSC browser. The expression heat map (Fig. 2C–D) and the following regression analysis (Fig. 2E–F) showed that the correlation co-efficiency between MASTL and E2F8 was higher in ER+ group than in ERgroup (Pearson’ r = 0.59 and 0.45 respectively, Fig. 2E–F). 3.3. E2F8 transcriptionally activates MASTL via binding to its promoter To further examine the possible mechanism underlying their co-expression, two ER+ breast cancer cell lines, MCF-7 and BT474 cells were firstly infected with E2F8 lentiviral particles (Fig. 3A). E2F8 overexpression significantly enhanced MASTL expression at both mRNA and protein level in both MCF-7 and BT474 cells (Fig. 3B–C). Promoter scanning using the JASPAR Database showed that the MASTL promoter region has a highly possible E2F8 binding site upstream (356 to 345) the TSS site (Fig. 3D). To verify the predicted binding site, we further generated several luciferase
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reporter plasmids carrying different truncated MASTL promoter sequences for dual luciferase assay. In HEK-293 cells, E2F8 overexpression significantly elevated transcription activity of the plasmids with intact E2F8 binding site (Fig. 3E). However, the effect was abrogated by deleting the predicted binding site (Fig. 3E). In order to further validate the direct binding of E2F8 to MASTL promoter, ChIP with following qRT-PCR were performed. The results confirmed that E2F8 can effectively bind to the MASTL promoter in both MCF-7 and BT474 cells (Fig. 3F). To further verify the trans-activating effect of E2F8 in ER+ breast cancer cells, the expression of CCNE1 and CCNE2, two known downstream targets of E2F8 in breast cancer [14] and CCND1, a known E2F8 target in hepatocellular carcinoma [21], were examined. Data mining in TCGA-BRCA showed that E2F8 is positive correlated to the expression of CCNE1 and CCNE2 (Pearson’s r = 0.60 and 0.71 respectively), but was not associated with CCND1 expression (Pearson’s r = 0.12) (Fig. 3G–H). In both MCF-7 and BT-474 cells, we confirmed that E2F8 overexpression induced upregulation of CCNE1 and CCNE2, but had no influence on CCND1 expression
Fig. 4. E2F8 upregulation is associated with poor prognosis in breast cancer. A–D. Kaplan-Meier plots of the association between E2F8 expression and DMFS (A–B) and RFS (C–D) in ER+ (A and C) or ER- (B and D) breast cancer patients. Data was obtained by using Kaplan-Meier Plotter.
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(Fig. 3I). These results suggest that the trans-activating effect of E2F8 might be cancer specific. 3.4. E2F8 upregulation is associated with poor prognosis in breast cancer To assess whether dysregulated E2F8 is associated with DMFS/ RFS in breast cancer patients, data mining was also performed using Kaplan-Meier Plotter. The survival curve showed that high E2F8 expression was significantly associated with worse DMFS (Hazard Ratio (HR): 2.99, 95%CI: 2.04–4.38; p < 0.001) and also worse RFS (HR): 2.00, 95%CI: 1.66–2.41; (p < 0.001) in ER+ breast cancer patients (Fig. 4A and C). However, no association between E2F8 expression and DMFS was observed in ER- patients (Fig. 4B). In addition, E2F8 upregulation is only associated with worse RFS in ER- patients at the margin level of significance (HR: 1.27, 95%CI: 1.01–1.6; p = 0.042) (Fig. 4D). 3.5. E2F8 shortens cisplatin induced G2/M arrest by MASTL mediated mitotic progression Since E2F8 is significantly associated with worse DMFS/RFS in ER+ breast cancer patients, we further investigated whether it is involved in drug responses in MCF-7 cells. The cells were firstly transfected with E2F8 or MASTL lentiviral particles or co-infected with E2F8 lentiviral particles and MASTL lentiviral shRNA (Fig. 5A). CCK-8 assay of cell viability showed that E2F8 and MASTL
overexpression significantly increased viability of MCF-7 cells after cisplatin treatment (Fig. 5B–C). In comparison, inhibition of MASTL significantly weakened the protective effect of E2F8 on maintaining cell viability after cisplatin treatment (Fig. 5D). By performing flow cytometric assay, we observed that E2F8 and MASTL overexpression significantly reduced cisplatin induced activation of caspase-9 (Fig. 5E–H). However, inhibition of MASTL significantly weakened the protective effect of E2F8 on cisplatin induced cell apoptosis (Fig. 5I–J). Then, we investigated the underlying mechanism. By monitoring cell cycle distribution, we observed that cisplatin induced G2/M arrest at 32 h after cisplatin treatment (Fig. 5K). In vector control group, majority of the cells continued to arrest over the next 16 h (Fig. 5K). In comparison, in E2F8 or MASTL overexpression group, over 40% cells recovered and entered G1 and S phase during this period (Fig. 5K). This transition was further validated by western blot analysis of accumulation of histone H3Ser10 phosphorylation (Fig. 5J), which indicates checkpoint recovery and mitotic entry [22,23]. In addition, by quantitation of the proportion of cells in sub-G1 phase, we also confirmed that E2F8 overexpression substantially reduced cisplatin induced cell death, while inhibition of MASTL partly abrogated the protective effect (Fig. 5L and M). 4. Discussion One recent study observed that MASTL upregulation was strongly correlated with poor overall survival in breast cancer
Fig. 5. E2F8 shortens cisplatin induced G2/M arrest by MASTL mediated mitotic progression. A. Western blotting of MASTL protein expression in MCF-7 cells 24 h after infection of lentiviral E2F8 or MASTL expression particles or co-infection of lentiviral E2F8 expression particles and lentiviral MASTL shRNA. B–D. 24 h after indicating infections in figure A, the cells were subjected to cisplatin treatment for another 48 h. Then, CCK-8 assay was performed to measure cell viability. E–J. 24 h after indicating infections in figure A, the cells were subjected to 5 mM cisplatin treatment for another 48 h. Representative flow cytometric images of cells with active caspase-9 after the treatment (E, G and I). Quantitation of the proportion of cells with active caspase-9 (F, H and J). K–M. 24 h after indicating infections in figure A, the cells were grown in the presence of cisplatin for 48 h. At the indicated time points, the cells were harvested for flow cytometric analysis of cell cycle distribution (K) or immunoblotting of histone H3Ser10 phosphorylation (L). At 48 h, the proportion of cells in sub-G1 phase in each groups were quantified (M). ** p < 0.01.
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patients [11]. In this study, by data mining in large databases, we further demonstrated that high MASTL expression was associated with worse DMFS and RFS in ER+ patient cancer patients. The oncogenic properties of MASTL in breast cancer cells have also been gradually revealed. Ectopic expression of MASTL significantly stimulated proliferation of head and neck squamous cancer cells under cisplatin-induced stress conditions [11]. However, the mechanism of its dysregulation is not clear. Our data showed that in TCGA-BRCA, MASTL and E2F8 are coupregulated. Mechanistically, E2F8 transcriptionally activates MASTL via binding to its promoter. In addition, we verified that E2F8 overexpression induced upregulation of CCNE1 and CCNE2, two known downstream targets of E2F8 in breast cancer [14], but had no influence on CCND1 expression, a known E2F8 target in hepatocellular carcinoma [21]. These results suggest that the trans-activating effect of E2F8 might be cancer specific. By data mining in Kaplan-Meier Plotter, we observed that high E2F8 expression is associated with worse DMFS and RFS in ER+ patients. Therefore, we hypothesized that E2F8 might exert oncogenic effect via MASTL in ER+ breast cancer. As other genotoxic drugs or radiation, cisplatin exerts cytotoxicity mainly via inducing DNA damage. Cisplatin treatment directly leads to activation of the checkpoint at G2/M phase, which subsequently leads to cell apoptosis to eliminate the damaged cells [4]. One recent study investigated cancer cell fate after cisplatin treatment and found that checkpoint slippage occurred predominantly in late S and G2 phases, while the cells in M-phase were hypersensitive to cisplatin [4]. Prolonged mitosis was correlated with the induction of cell death [4]. Therefore, the genes modulating cell cycle progression might also affect cisplatin sensitivity. Previous studies showed that both E2F8 and MASTL are regulator in cell cycle progression. Although E2F8 was initially identified as a transcription suppressor, emerging evidence suggest that it can function as either transcription activators or repressors, depending on the cellular and tissue context, or the target genes [14]. For example, E2F8 can transcriptionally upregulate cyclin D1 in hepatocellular carcinoma [21], and UHRF1 in lung cancer [24]. E2F8 can facilitate G1/S transition in breast cancer cells via transcriptionally activating the expression of CCNE1 and CCNE2 [14]. Inhibition of E2F8 was sufficient to suppress prostate cancer cell growth by inducing G2/M arrest [15]. MASTL in human is a key player for mitosis. Functionally, MASTL can phosphorylate its substratesa-endosulfine (ENSA) and/or cAMP-regulated phosphoprotein 19 (ARPP19). After phosphorylation, ENSA and ARPP19 inactivate the protein phosphatase 2A complex (PP2A/B55) [25], which is the principal protein phosphatase complex that dephosphorylates CDK substrates, including CDK1 [25,26]. This helps to maintain CDK1 activity above the threshold required for mitotic entry [8]. In in vitro IR induced cell DNA damage model, one recent study found that although MASTL had no influence on activation and inactivation of the DNA damage checkpoint, it modulated the speed of mitotic entry after DNA damage via its regulative effect on CDK1 inhibitory phosphorylation [3]. In the absence of MASTL, DNA-damaged cells showed delayed mitotic entry after DNA damage and could not undergo genome re-duplication [3]. In contrast, MASTL overexpression accelerated mitotic entry after DNA damage [3]. Considering the regulative effect of E2F8 on MASTL expression, we also examined whether E2F8 modulates cisplatin sensitivity of ER+ breast cancer cells via MASTL. Our results showed that E2F8 overexpression alleviated cisplatin induced cell apoptosis in MCF-7 cells by shortening G2/M arrest and promoting mitotic entry, the effect of which was largely canceled by inhibiting MASTL.
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5. Conclusion Based on the findings, we infer that E2F8 can shorten cisplatin induced G2/M arrest by promoting MASTL mediated mitotic progression in ER+ breast cancer cells. These findings might help to explain why high MASTL or high E2F8 expression is associated with worse RFS in ER+ breast cancer. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. biopha.2017.05.118. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics 2014, CA: Cancer J. Clin. 64 (1) (2014) 9–29. [2] D.X. He, F. Gu, F. Gao, J.J. Hao, D. Gong, X.T. Gu, A.Q. Mao, J. Jin, L. Fu, X. Ma, Genome-wide profiles of methylation, microRNAs, and gene expression in chemoresistant breast cancer, Sci. Rep. 6 (2016) 24706. [3] P.Y. Wong, H.T. Ma, H.J. Lee, R.Y. Poon, MASTL(greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation, Sci. Rep. 6 (2016) 22230. [4] K.V. Luong, L. Wang, B.J. Roberts, J.K. Wahl 3rd, A. Peng, Cell fate determination in cisplatin resistance and chemosensitization, Oncotarget 7 (17) (2016) 23383–23394. [5] L. Cali Cassi, G. Vanni, G. Petrella, P. Orsaria, C. Pistolese, G. Lo Russo, M. Innocenti, O. Buonomo, Comparative study of oncoplastic versus nononcoplastic breast conserving surgery in a group of 211 breast cancer patients, Eur. Rev. Med. Pharmacol. Sci. 20 (14) (2016) 2950–2954. [6] M.L. Xing, Y.F. Lu, D.F. Wang, X.Y. Zou, S.X. Zhang, Z. Yun, Clinical significance of sCIP2A levels in breast cancer, Eur. Rev. Med. Pharmacol. Sci. 20 (1) (2016) 82– 91. [7] E. Voets, R. Wolthuis, MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C, Biol. Open 4 (4) (2015) 484–495. [8] C.A. Ocasio, M.B. Rajasekaran, S. Walker, D. Le Grand, J. Spencer, F.M. Pearl, S.E. Ward, V. Savic, L.H. Pearl, H. Hochegger, A.W. Oliver, A first generation inhibitor of human greatwall kinase, enabled by structural and functional characterisation of a minimal kinase domain construct, Oncotarget 7 (44) (2016) 71182–71197. [9] A. Peng, T.M. Yamamoto, M.L. Goldberg, J.L. Maller, A novel role for greatwall kinase in recovery from DNA damage, Cell cycle 9 (21) (2010) 4364–4369. [10] N. Motoyama, K. Naka, DNA damage tumor suppressor genes and genomic instability, Curr. Opin. Genet. Dev. 14 (1) (2004) 11–16. [11] L. Wang, V.Q. Luong, P.J. Giannini, A. Peng, Mastl kinase, a promising therapeutic target, promotes cancer recurrence, Oncotarget 5 (22) (2014) 11479–11489. [12] H.Z. Chen, M.M. Ouseph, J. Li, T. Pecot, V. Chokshi, L. Kent, S. Bae, M. Byrne, C. Duran, G. Comstock, P. Trikha, M. Mair, S. Senapati, C.K. Martin, S. Gandhi, N. Wilson, B. Liu, Y.W. Huang, J.C. Thompson, S. Raman, S. Singh, M. Leone, R. Machiraju, K. Huang, X. Mo, S. Fernandez, I. Kalaszczynska, D.J. Wolgemuth, P. Sicinski, T. Huang, V. Jin, G. Leone, Canonical and atypical E2Fs regulate the mammalian endocycle, Nat. Cell Biol. 14 (11) (2012) 1192–1202. [13] E. Petru, F. Moinfar, S. Graf, Regulation of the E2F family: a further step in understanding ovarian cancer biology, Cancer. Biol. Ther. 5 (7) (2006) 777–778. [14] L. Ye, L. Guo, Z. He, X. Wang, C. Lin, X. Zhang, S. Wu, Y. Bao, Q. Yang, L. Song, H. Lin, Upregulation of E2F8 promotes cell proliferation and tumorigenicity in breast cancer by modulating G1/S phase transition, Oncotarget 7 (17) (2016) 23757–23771. [15] S. Lee, Y.R. Park, S.H. Kim, E.J. Park, M.J. Kang, I. So, J.N. Chun, J.H. Jeon, Geraniol suppresses prostate cancer growth through down-regulation of E2F8, Cancer Med. 5 (10) (2016) 2899–2908. [16] S. Tabaries, V. Ouellet, B.E. Hsu, M.G. Annis, A.A. Rose, L. Meunier, E. Carmona, C.E. Tam, A.M. Mes-Masson, P.M. Siegel, Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases, Breast Cancer Res. 17 (2015) 45. [17] A.M. Szasz, A. Lanczky, A. Nagy, S. Forster, K. Hark, J.E. Green, A. Boussioutas, R. Busuttil, A. Szabo, B. Gyorffy, Cross-validation of survival associated biomarkers in gastric cancer using transcriptomic data of 1,065 patients, Oncotarget 7 (31) (2016) 49322–49333. [18] B. Gyorffy, A. Lanczky, A.C. Eklund, C. Denkert, J. Budczies, Q. Li, Z. Szallasi, An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients, Breast Cancer Res. Treat. 123 (3) (2010) 725–731. [19] E. Cerami, J. Gao, U. Dogrusoz, B.E. Gross, S.O. Sumer, B.A. Aksoy, A. Jacobsen, C. J. Byrne, M.L. Heuer, E. Larsson, Y. Antipin, B. Reva, A.P. Goldberg, C. Sander, N. Schultz, The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data, Cancer Discov. 2 (5) (2012) 401–404.
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Original article
E2F8 confers cisplatin resistance to ER+ breast cancer cells via transcriptionally activating MASTL Jianjun Tiana,1, Yuting Linb,1, Jianhua Yuc,* a
Clinical Laboratory, Qilu Children’s Hospital of Shandong University, Jinan, 250000, Shandong, China Clinical Laboratory, Beihai Hospital of Yantai, Yantai, 264000, Shandong, China c Clinical Laboratory, The 148th Hospital of Chinese People’s Liberation Army, Zibo, 255000, Shandong, China b
A R T I C L E I N F O
Article history: Received 12 April 2017 Received in revised form 15 May 2017 Accepted 24 May 2017 Keywords: E2F8 Cisplatin resistance Breast cancer MASTL Cell cycle
A B S T R A C T
MASTL (microtubule-associated serine/threonine kinase-like) is a critical kinase modulating mitotic entry. In this study, we investigated the mechanism of its dysregulation in breast cancer and its involvement in cisplatin resistance in ER+ breast cancer cells. Data mining in Kaplan-Meier Plotter showed that high MASTL expression was associated with worse distant metastasis free survival (DMFS) and relapse free survival (RFS) in ER+ breast cancer patients. In TCGA breast cancer cohort (TCGA-BRCA), MASTL was strongly co-upregulated with E2F8. High E2F8 expression was also strongly associated with unfavorable DMFS and RFS in ER+ breast cancer patients. Promoter scanning in JASPAR Database showed that the MASTL promoter region has a highly possible E2F8 binding site upstream the TSS site. The following western blot, dual luciferase assay and ChIP-qPCR validated this binding site. In MCF-7 cells, E2F8 overexpression alleviated cisplatin induced cell apoptosis by shortening G2/M arrest and promoting mitotic entry, the effect of which was largely canceled by inhibiting MASTL. Therefore, we infer that E2F8 can shorten cisplatin induced G2/M arrest by promoting MASTL mediated mitotic progression in ER+ breast cancer cells. These findings might help to explain why high MASTL or high E2F8 expression is associated with worse RFS in ER+ breast cancer. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Breast cancer is the leading cancer in women across the world [1]. For the patients with advanced breast cancer, chemotherapy is still the major therapeutic strategy [2,3]. Cisplatin, together with other platinum-based drugs, are most successful and commonly used chemotherapeutic drugs [4]. However, the tumors initially responsive to chemotherapeutic agents usually develop acquired drug resistance after several rounds of therapy, which is the major cause of relapse, metastasis and finally cancer related death [5,6]. MASTL (microtubule-associated serine/threonine kinase-like) is a critical kinase modulating mitotic entry. In brief, MASTL can be activated directly by cyclin B-Cdk1 [7]. Once activated, MASTL supports further phosphorylation of cyclin B-Cdk1 substrates by restraining PP2A [7]. This feedback loop directly boosts CDK1 activity above the threshold required for mitotic entry [8]. One recent study reported that MASTL also acts as a regulator of the
* Corresponding author. E-mail address: [email protected] (J. Yu). Jianjun Tian and Yuting Lin contributed equally to this study.
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http://dx.doi.org/10.1016/j.biopha.2017.05.118 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
DNA damage response (DDR), a cellular surveillance mechanism [9]. In fact, the chemotherapeutic drugs are usually DNA damaging agents that activate DDR and following DNA repair, cell cycle checkpoint, and cell death [10]. Therefore, it is generally recognized that the dysregulated genes related to DDR are critically involved in cancer progression and therapy. In breast cancer, MASTL overexpression induces oncogenic properties such as transformation and invasiveness of the cancer cells [8]. In breast cancer, MASTL upregulation is correlated with a more advanced clinical stage [11]. However, the mechanism underlying its dysregulation in breast cancer is not clear. The E2Fs are a family of transcription factors that can bind to target promoters and regulate their transcription [12]. Traditionally, E2F1-E2F3 are considered as transcription activators, while E2F4E2F8 act as repressors [13]. However, in breast cancer, one recent study found that E2F8 acts as a transcription activator that upregulates the expression of CCNE1 and CCNE2 via directly interacting with their respective gene promoter, thereby promoting cancer cell proliferation [14]. Another recent study reported that in prostate cancer, knockdown of E2F8 was sufficient to suppress cell growth by inducing G2/M arrest [15]. These findings suggest that E2F8 might be an important gene modulating cell cycle progression.
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In this study, we found that E2F8 can transcriptionally activate MASTL via binding to its promoter and this regulative axis shortens cisplatin induced G2/M arrest in ER+ breast cancer cells. 2. Materials and methods 2.1. Microarray reanalysis The raw microarray data (GDS5666) [16] that compared gene expression profiles among the parental mammary tumor cell line 4T1 and 4T1 derived metastatic populations isolated from liver, lung or bone were downloaded from GEO datasets. The raw data of bone-aggressive explant and primary tumor explant were reanalyzed to identify the dysregulated genes. 2.2. Bioinformatic data mining To pool available annotated genomic data that assessed the association between MASTL/E2F8 expression and distant metastasis free survival (DMFS) and relapse-free survival (RFS) in breast cancer patients, data mining was performed using Kaplan-Meier Plotter (http://kmplot.com/analysis/), an online database that provides assessment of the effect of 54,675 genes on survival using 10,293 cancer samples, including 22,277 genes in 5143 breast cancer samples [17,18]. The survival curve were generated by using the JetSet best probe set with automatically selected best cutoff. The genes co-expressed with MASTL in TCGA breast cancer cohort (TCGA-BRCA) were identified using cBioPortal for Cancer
Genomics (http://cbioportal.org) [19]. The heat map of MASTL and E2F8, and their correlation were further studied using UCSC Xena Browser (http://xena.ucsc.edu/). The promoter sequence of MASTL was obtained from GeneCopoeia (> HPRM37364, NM_001172303) and was given in Supplementary material 1. The possible E2F8 binding sites in MASTL promoter region were predicted by using the JASPAR Database (http://jaspar.genereg.net/). The TSS site and the predicted E2F8 binding sites were marked in red font in Supplementary material 1. 2.3. Cell culture and transfection The human ER+ breast cancer cell line MCF-7 and BT474 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 10 units/mL penicillin, and 10 mg/mL streptomycin. MASTL Human Lentifect purified lentiviral particles (NM_001172303.1) and E2F8 Human ORFeome purified lentiviral particles, and lentiviral MASTL shRNA (HSH067101-LVRH1GP, based on psi-LVRH1GP vector) were obtained from Genecopoeia. Cells were infected with the lentiviral particles in the presence of 5 mg/mL polybrene. 2.4. Western blot analysis Conventional western blotting was performed as described previously [20]. Antibodies against E2F8, phosphor-histone
Fig. 1. High MASTL expression is associated with worse DMFS and RFS in ER+ breast cancer. A. The heap map the 30 most upregulated genes in 4T1 derived bone-aggressive explants compared to primary tumor. Red: up-regulation; Blue: down-regulation. Raw microarray data was obtained from GEO dataset (accession: GDS5666). B–E. Kaplan-Meier plots of the association between MASTL expression and DMFS (B–C) and RFS (D-E) in ER+ (B and D) or ER- (C and E) breast cancer patients. Data was obtained by using Kaplan-Meier Plotter.
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H3Ser10, MASTL, CCNE1, CCND2, CCND1 and b-actin were obtained from Abcam (Cambridge, MA, USA). The protein bands were visualized by using the SuperSignalTM West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, lL, USA).
system with a luminometer (Promega, Madison, WI, USA) following the manufacturer’s instructions.
2.5. QRT-PCR
ChIPassaywasperformedbyusingtheUpstate-ChIPAssayKit(Lake Placid, NY, USA) following the manufacturer's instructions. Equal aliquots of chromatin supernatants from MCF-7 cells were subjected to immunoprecipitation with 1 mg of normal goat IgG or anti-E2F8 (ab109596,Abcam)overnightat4 Cwithrotation.Afterreversecrosslink of protein/DNA complexes to free DNA, the ChIP-enriched DNA was analyzed by qRT-PCR using the ABI 7900HT sequence detection system and SYBR green master mix. Primers used were: GAPDH, 50 agcgcaggcctcaagacctt-'3 (forward) and 50 -aagaagatgcggctgactgt-30 (reverse), and MASTL promoter, 50 -gacggagtttcgttctttcg-30 (forward) and 50 -ggcccagtcagaagatttca-30 (reverse).
24 h after infection of lentiviral E2F8 expression particles or the empty control, MCF-7 and BT474 cells were lysed for exaction of total RNA, with the using of Trizol Reagent (Invitrogen). Then, cDNA was obtained by reverse transcription using a First-Strand Synthesis Kit (Invitrogen). To detect E2F8 mRNA expression, qRTPCR analysis was performed on an ABI 7900HT sequence detector using gene specific primers (E2F8, forward: 50 -CCAACCCTGCTGTGAATA-30 ; reverse: 50 -TTTCTGGCTCATTACCCT-30 ) and TaqMan Universal PCR Master Mix according to the directions of manufacturer (Applied Biosystems, Foster City, CA, USA). The 2DDCt method was used to calculate the relative expression. 2.6. Dual luciferase assay MASTL promoter fragments (1309 to +173, 1000 to +173, 700 to +173, 400 to +173, 300 to +173 and 100 to +173) were PCR amplified from the MASTL promoter clone (HPRM37364) and then were inserted into the XhoI-Hind III site of the pGL3-basic luciferase reporter vector respectively. Then, dual luciferase assay was performed as described previously [20]. In brief, HEK-293 cells were seeded into 12-well plates at a density of 2 105 cells per well and were further infected with lentiviral E2F8 expression particles or the empty control. 24 h later, the cells were co-transfected with 1.5 mg luciferase construct plasmid or the empty reporter vector DNA and 0.05 mg phRL-TK by using Superfectin (Qiagen, Valencia, CA, USA). 24 h later, the cells were lyzed and luciferase activity of the lysate was measured using the dual-luciferase reporter assay
2.7. Chromatin immunoprecipitation assay (ChIP)
2.8. Flow cytometric analysis of active caspase-9 24 h after infection, MCF-7 cells were subjected to 5 mM cisplatin treatment for another 48 h. After the treatment, the proportion of cells with activated caspase-9 was determined using the GaspGLOW TM Fluorescein Active Caspase-9 Staining Kit (Biovision, Mountain View, CA, USA) according to the manufacturer’s protocol. Fluorescence was excited with the 488-nm line of an argon laser on an EPICS XL flowcytometer (Beckman Coulter, Fullerton, CA, USA). 2.9. Cell cycle analysis MCF-7 after indicating treatment and at indicating time points were collected, washed and fixed with ice-cold methanol. Then the cells were stained with the Cellometer PI Cell Cycle Kit (Nexcelom Bioscience, Lawrence, MA, USA) according to the protocol
Fig. 2. MASTL and E2F8 are co-upregulated in breast cancer. A. The top genes positively correlated to MASTL expression in TCGA-BRCA. B. The correlation between MASTL and E2F8 in TCGA-BRCA. Analysis was performed by using cBioPortal. C–F. The heat map (C–D) and regression analysis (E–F) of the expression between MASTL and E2F8 in ER+ and ER- subgroups in TCGA-BRCA. Analysis was performed using UCSC Xena browser.
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recommended by the manufacturer. After that, the cells were loaded into the cellometer counting chamber and the cell cycle distribution was determined using Partec flow cytometer.
3. Results 3.1. High MASTL expression is associated with worse DMFS and RFS in ER+ breast cancer
2.10. CCK-8 assay of cell viability 24 h after indicating transfection, MCF-7 cells were seeded in a 96-well plate and were further treated with varying concentrations of cisplatin for 48 h. After that, cell viability was measured using the Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to manufacturer’s instruction. In brief, CCK-8 solution was added to each well and the plate was placed in a cell incubator for 4 h. Cell viability was reflected by the absorbance at 450 nm determined by a 96-well spectrophotometry. 2.11. Statistical analysis Data were presented in the form of means standard deviation (SD) with data from three repeats. Data were analyzed for statistical significance by two-tailed Student’s t-test or ANOVA with Student-Newman-Keuls test as a post hoc test. p value of <0.05 was considered statistically significant.
One recent study reported that MASTL has oncogenic properties in breast cancer and its upregulation is associated with cancer progression [11]. By reanalysis of the raw data of one previous microarray (GDS5666), we observed that MASTL upregulation is significantly associated with bone metastasis compared to primary tumor (Fig. 1A, red arrow). Then, we explored whether there is any association between MASTL expression and survival outcomes in breast cancer patients by data mining in Kaplan-Meier Plotter. The results showed that high MASTL expression was associated with worse DMFS and RFS in ER+ breast cancer patients (Fig. 1B and D), but not in ER- patients (Fig. 1C and E). 3.2. MASTL and E2F8 are co-upregulated in breast cancer Then, we tried to explore the underlying mechanism of MASTL dysregulation in breast cancer. By data mining in TCGA-BRCA using the cBioPortal, we found that E2F8 was positively correlated with
Fig. 3. E2F8 transcriptionally activates MASTL via binding to its promoter. A. Western blot analysis of E2F8 expression in MCF-7 and BT474 cells 24 h after infection of lentiviral E2F8 expression particles or the empty control. B-C. QRT-PCR analysis of MASTL mRNA (B) and western blotting of MASTL protein (C) expression in MCF-7 and BT474 cells 36 h after infection of lentiviral E2F8 expression particles or the empty control. D. Predicted E2F8 binding site in MASTL promoter. E. The luciferase reporter constructs carrying truncated MASTL promoter sequences were introduced into HEK-293 cells pre-infected with lentiviral E2F8 expression particles or the empty control. Luciferase activity was measured 24 h post-transfection. F. Fold-enrichment of E2F8 binding at MASTL promoter relative to background in MCF-7 cells was measured by CHIP-qPCR. Upon normalization to GAPDH, results were expressed as n-fold compared to IgG. G. The heat map of E2F8, CCNE1, CCNE2 and CCND1 expression in ER+ subgroup in TCGA-BRCA. H. Summary of regression analysis of the correlation between E2F8 and CCNE1, between E2F8 and CCNE2 and between E2F8 and CCND1. I. Western blotting of E2F8, CCNE1, CCNE2 and CCND1 expression in MCF-7 and BT474 cells 36 h after infection of lentiviral E2F8 expression particles or the empty control. **, p < 0.01.
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MASTL expression (Pearson’ r = 0.66, Fig. 2A–B). To further verify the co-expression in ER+ and ER- patients, their expression profiles in TCGA-BRCA were also checked using the UCSC browser. The expression heat map (Fig. 2C–D) and the following regression analysis (Fig. 2E–F) showed that the correlation co-efficiency between MASTL and E2F8 was higher in ER+ group than in ERgroup (Pearson’ r = 0.59 and 0.45 respectively, Fig. 2E–F). 3.3. E2F8 transcriptionally activates MASTL via binding to its promoter To further examine the possible mechanism underlying their co-expression, two ER+ breast cancer cell lines, MCF-7 and BT474 cells were firstly infected with E2F8 lentiviral particles (Fig. 3A). E2F8 overexpression significantly enhanced MASTL expression at both mRNA and protein level in both MCF-7 and BT474 cells (Fig. 3B–C). Promoter scanning using the JASPAR Database showed that the MASTL promoter region has a highly possible E2F8 binding site upstream (356 to 345) the TSS site (Fig. 3D). To verify the predicted binding site, we further generated several luciferase
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reporter plasmids carrying different truncated MASTL promoter sequences for dual luciferase assay. In HEK-293 cells, E2F8 overexpression significantly elevated transcription activity of the plasmids with intact E2F8 binding site (Fig. 3E). However, the effect was abrogated by deleting the predicted binding site (Fig. 3E). In order to further validate the direct binding of E2F8 to MASTL promoter, ChIP with following qRT-PCR were performed. The results confirmed that E2F8 can effectively bind to the MASTL promoter in both MCF-7 and BT474 cells (Fig. 3F). To further verify the trans-activating effect of E2F8 in ER+ breast cancer cells, the expression of CCNE1 and CCNE2, two known downstream targets of E2F8 in breast cancer [14] and CCND1, a known E2F8 target in hepatocellular carcinoma [21], were examined. Data mining in TCGA-BRCA showed that E2F8 is positive correlated to the expression of CCNE1 and CCNE2 (Pearson’s r = 0.60 and 0.71 respectively), but was not associated with CCND1 expression (Pearson’s r = 0.12) (Fig. 3G–H). In both MCF-7 and BT-474 cells, we confirmed that E2F8 overexpression induced upregulation of CCNE1 and CCNE2, but had no influence on CCND1 expression
Fig. 4. E2F8 upregulation is associated with poor prognosis in breast cancer. A–D. Kaplan-Meier plots of the association between E2F8 expression and DMFS (A–B) and RFS (C–D) in ER+ (A and C) or ER- (B and D) breast cancer patients. Data was obtained by using Kaplan-Meier Plotter.
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(Fig. 3I). These results suggest that the trans-activating effect of E2F8 might be cancer specific. 3.4. E2F8 upregulation is associated with poor prognosis in breast cancer To assess whether dysregulated E2F8 is associated with DMFS/ RFS in breast cancer patients, data mining was also performed using Kaplan-Meier Plotter. The survival curve showed that high E2F8 expression was significantly associated with worse DMFS (Hazard Ratio (HR): 2.99, 95%CI: 2.04–4.38; p < 0.001) and also worse RFS (HR): 2.00, 95%CI: 1.66–2.41; (p < 0.001) in ER+ breast cancer patients (Fig. 4A and C). However, no association between E2F8 expression and DMFS was observed in ER- patients (Fig. 4B). In addition, E2F8 upregulation is only associated with worse RFS in ER- patients at the margin level of significance (HR: 1.27, 95%CI: 1.01–1.6; p = 0.042) (Fig. 4D). 3.5. E2F8 shortens cisplatin induced G2/M arrest by MASTL mediated mitotic progression Since E2F8 is significantly associated with worse DMFS/RFS in ER+ breast cancer patients, we further investigated whether it is involved in drug responses in MCF-7 cells. The cells were firstly transfected with E2F8 or MASTL lentiviral particles or co-infected with E2F8 lentiviral particles and MASTL lentiviral shRNA (Fig. 5A). CCK-8 assay of cell viability showed that E2F8 and MASTL
overexpression significantly increased viability of MCF-7 cells after cisplatin treatment (Fig. 5B–C). In comparison, inhibition of MASTL significantly weakened the protective effect of E2F8 on maintaining cell viability after cisplatin treatment (Fig. 5D). By performing flow cytometric assay, we observed that E2F8 and MASTL overexpression significantly reduced cisplatin induced activation of caspase-9 (Fig. 5E–H). However, inhibition of MASTL significantly weakened the protective effect of E2F8 on cisplatin induced cell apoptosis (Fig. 5I–J). Then, we investigated the underlying mechanism. By monitoring cell cycle distribution, we observed that cisplatin induced G2/M arrest at 32 h after cisplatin treatment (Fig. 5K). In vector control group, majority of the cells continued to arrest over the next 16 h (Fig. 5K). In comparison, in E2F8 or MASTL overexpression group, over 40% cells recovered and entered G1 and S phase during this period (Fig. 5K). This transition was further validated by western blot analysis of accumulation of histone H3Ser10 phosphorylation (Fig. 5J), which indicates checkpoint recovery and mitotic entry [22,23]. In addition, by quantitation of the proportion of cells in sub-G1 phase, we also confirmed that E2F8 overexpression substantially reduced cisplatin induced cell death, while inhibition of MASTL partly abrogated the protective effect (Fig. 5L and M). 4. Discussion One recent study observed that MASTL upregulation was strongly correlated with poor overall survival in breast cancer
Fig. 5. E2F8 shortens cisplatin induced G2/M arrest by MASTL mediated mitotic progression. A. Western blotting of MASTL protein expression in MCF-7 cells 24 h after infection of lentiviral E2F8 or MASTL expression particles or co-infection of lentiviral E2F8 expression particles and lentiviral MASTL shRNA. B–D. 24 h after indicating infections in figure A, the cells were subjected to cisplatin treatment for another 48 h. Then, CCK-8 assay was performed to measure cell viability. E–J. 24 h after indicating infections in figure A, the cells were subjected to 5 mM cisplatin treatment for another 48 h. Representative flow cytometric images of cells with active caspase-9 after the treatment (E, G and I). Quantitation of the proportion of cells with active caspase-9 (F, H and J). K–M. 24 h after indicating infections in figure A, the cells were grown in the presence of cisplatin for 48 h. At the indicated time points, the cells were harvested for flow cytometric analysis of cell cycle distribution (K) or immunoblotting of histone H3Ser10 phosphorylation (L). At 48 h, the proportion of cells in sub-G1 phase in each groups were quantified (M). ** p < 0.01.
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patients [11]. In this study, by data mining in large databases, we further demonstrated that high MASTL expression was associated with worse DMFS and RFS in ER+ patient cancer patients. The oncogenic properties of MASTL in breast cancer cells have also been gradually revealed. Ectopic expression of MASTL significantly stimulated proliferation of head and neck squamous cancer cells under cisplatin-induced stress conditions [11]. However, the mechanism of its dysregulation is not clear. Our data showed that in TCGA-BRCA, MASTL and E2F8 are coupregulated. Mechanistically, E2F8 transcriptionally activates MASTL via binding to its promoter. In addition, we verified that E2F8 overexpression induced upregulation of CCNE1 and CCNE2, two known downstream targets of E2F8 in breast cancer [14], but had no influence on CCND1 expression, a known E2F8 target in hepatocellular carcinoma [21]. These results suggest that the trans-activating effect of E2F8 might be cancer specific. By data mining in Kaplan-Meier Plotter, we observed that high E2F8 expression is associated with worse DMFS and RFS in ER+ patients. Therefore, we hypothesized that E2F8 might exert oncogenic effect via MASTL in ER+ breast cancer. As other genotoxic drugs or radiation, cisplatin exerts cytotoxicity mainly via inducing DNA damage. Cisplatin treatment directly leads to activation of the checkpoint at G2/M phase, which subsequently leads to cell apoptosis to eliminate the damaged cells [4]. One recent study investigated cancer cell fate after cisplatin treatment and found that checkpoint slippage occurred predominantly in late S and G2 phases, while the cells in M-phase were hypersensitive to cisplatin [4]. Prolonged mitosis was correlated with the induction of cell death [4]. Therefore, the genes modulating cell cycle progression might also affect cisplatin sensitivity. Previous studies showed that both E2F8 and MASTL are regulator in cell cycle progression. Although E2F8 was initially identified as a transcription suppressor, emerging evidence suggest that it can function as either transcription activators or repressors, depending on the cellular and tissue context, or the target genes [14]. For example, E2F8 can transcriptionally upregulate cyclin D1 in hepatocellular carcinoma [21], and UHRF1 in lung cancer [24]. E2F8 can facilitate G1/S transition in breast cancer cells via transcriptionally activating the expression of CCNE1 and CCNE2 [14]. Inhibition of E2F8 was sufficient to suppress prostate cancer cell growth by inducing G2/M arrest [15]. MASTL in human is a key player for mitosis. Functionally, MASTL can phosphorylate its substratesa-endosulfine (ENSA) and/or cAMP-regulated phosphoprotein 19 (ARPP19). After phosphorylation, ENSA and ARPP19 inactivate the protein phosphatase 2A complex (PP2A/B55) [25], which is the principal protein phosphatase complex that dephosphorylates CDK substrates, including CDK1 [25,26]. This helps to maintain CDK1 activity above the threshold required for mitotic entry [8]. In in vitro IR induced cell DNA damage model, one recent study found that although MASTL had no influence on activation and inactivation of the DNA damage checkpoint, it modulated the speed of mitotic entry after DNA damage via its regulative effect on CDK1 inhibitory phosphorylation [3]. In the absence of MASTL, DNA-damaged cells showed delayed mitotic entry after DNA damage and could not undergo genome re-duplication [3]. In contrast, MASTL overexpression accelerated mitotic entry after DNA damage [3]. Considering the regulative effect of E2F8 on MASTL expression, we also examined whether E2F8 modulates cisplatin sensitivity of ER+ breast cancer cells via MASTL. Our results showed that E2F8 overexpression alleviated cisplatin induced cell apoptosis in MCF-7 cells by shortening G2/M arrest and promoting mitotic entry, the effect of which was largely canceled by inhibiting MASTL.
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5. Conclusion Based on the findings, we infer that E2F8 can shorten cisplatin induced G2/M arrest by promoting MASTL mediated mitotic progression in ER+ breast cancer cells. These findings might help to explain why high MASTL or high E2F8 expression is associated with worse RFS in ER+ breast cancer. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. biopha.2017.05.118. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics 2014, CA: Cancer J. Clin. 64 (1) (2014) 9–29. [2] D.X. He, F. Gu, F. Gao, J.J. Hao, D. Gong, X.T. Gu, A.Q. Mao, J. Jin, L. Fu, X. Ma, Genome-wide profiles of methylation, microRNAs, and gene expression in chemoresistant breast cancer, Sci. Rep. 6 (2016) 24706. [3] P.Y. Wong, H.T. Ma, H.J. Lee, R.Y. Poon, MASTL(greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation, Sci. Rep. 6 (2016) 22230. [4] K.V. Luong, L. Wang, B.J. Roberts, J.K. Wahl 3rd, A. Peng, Cell fate determination in cisplatin resistance and chemosensitization, Oncotarget 7 (17) (2016) 23383–23394. [5] L. Cali Cassi, G. Vanni, G. Petrella, P. Orsaria, C. Pistolese, G. Lo Russo, M. Innocenti, O. Buonomo, Comparative study of oncoplastic versus nononcoplastic breast conserving surgery in a group of 211 breast cancer patients, Eur. Rev. Med. Pharmacol. Sci. 20 (14) (2016) 2950–2954. [6] M.L. Xing, Y.F. Lu, D.F. Wang, X.Y. Zou, S.X. Zhang, Z. Yun, Clinical significance of sCIP2A levels in breast cancer, Eur. Rev. Med. Pharmacol. Sci. 20 (1) (2016) 82– 91. [7] E. Voets, R. Wolthuis, MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C, Biol. Open 4 (4) (2015) 484–495. [8] C.A. Ocasio, M.B. Rajasekaran, S. Walker, D. Le Grand, J. Spencer, F.M. Pearl, S.E. Ward, V. Savic, L.H. Pearl, H. Hochegger, A.W. Oliver, A first generation inhibitor of human greatwall kinase, enabled by structural and functional characterisation of a minimal kinase domain construct, Oncotarget 7 (44) (2016) 71182–71197. [9] A. Peng, T.M. Yamamoto, M.L. Goldberg, J.L. Maller, A novel role for greatwall kinase in recovery from DNA damage, Cell cycle 9 (21) (2010) 4364–4369. [10] N. Motoyama, K. Naka, DNA damage tumor suppressor genes and genomic instability, Curr. Opin. Genet. Dev. 14 (1) (2004) 11–16. [11] L. Wang, V.Q. Luong, P.J. Giannini, A. Peng, Mastl kinase, a promising therapeutic target, promotes cancer recurrence, Oncotarget 5 (22) (2014) 11479–11489. [12] H.Z. Chen, M.M. Ouseph, J. Li, T. Pecot, V. Chokshi, L. Kent, S. Bae, M. Byrne, C. Duran, G. Comstock, P. Trikha, M. Mair, S. Senapati, C.K. Martin, S. Gandhi, N. Wilson, B. Liu, Y.W. Huang, J.C. Thompson, S. Raman, S. Singh, M. Leone, R. Machiraju, K. Huang, X. Mo, S. Fernandez, I. Kalaszczynska, D.J. Wolgemuth, P. Sicinski, T. Huang, V. Jin, G. Leone, Canonical and atypical E2Fs regulate the mammalian endocycle, Nat. Cell Biol. 14 (11) (2012) 1192–1202. [13] E. Petru, F. Moinfar, S. Graf, Regulation of the E2F family: a further step in understanding ovarian cancer biology, Cancer. Biol. Ther. 5 (7) (2006) 777–778. [14] L. Ye, L. Guo, Z. He, X. Wang, C. Lin, X. Zhang, S. Wu, Y. Bao, Q. Yang, L. Song, H. Lin, Upregulation of E2F8 promotes cell proliferation and tumorigenicity in breast cancer by modulating G1/S phase transition, Oncotarget 7 (17) (2016) 23757–23771. [15] S. Lee, Y.R. Park, S.H. Kim, E.J. Park, M.J. Kang, I. So, J.N. Chun, J.H. Jeon, Geraniol suppresses prostate cancer growth through down-regulation of E2F8, Cancer Med. 5 (10) (2016) 2899–2908. [16] S. Tabaries, V. Ouellet, B.E. Hsu, M.G. Annis, A.A. Rose, L. Meunier, E. Carmona, C.E. Tam, A.M. Mes-Masson, P.M. Siegel, Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases, Breast Cancer Res. 17 (2015) 45. [17] A.M. Szasz, A. Lanczky, A. Nagy, S. Forster, K. Hark, J.E. Green, A. Boussioutas, R. Busuttil, A. Szabo, B. Gyorffy, Cross-validation of survival associated biomarkers in gastric cancer using transcriptomic data of 1,065 patients, Oncotarget 7 (31) (2016) 49322–49333. [18] B. Gyorffy, A. Lanczky, A.C. Eklund, C. Denkert, J. Budczies, Q. Li, Z. Szallasi, An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients, Breast Cancer Res. Treat. 123 (3) (2010) 725–731. [19] E. Cerami, J. Gao, U. Dogrusoz, B.E. Gross, S.O. Sumer, B.A. Aksoy, A. Jacobsen, C. J. Byrne, M.L. Heuer, E. Larsson, Y. Antipin, B. Reva, A.P. Goldberg, C. Sander, N. Schultz, The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data, Cancer Discov. 2 (5) (2012) 401–404.
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