ZNF500 level is elevated in breast cancer but negatively correlates with advanced TNM stage, positive lymph node metastasis, and poor prognosis
First, we performed bioinformatic analysis to explore the expression of ZNF500 in human malignancies. According to the Cancer Genome Atlas database pan-cancer analysis, ZNF500 mRNA expression was significantly elevated in most cancerous tissues compared to that in adjacent normal tissues (Figure 1A). We explored its expression specifically in breast cancer and found that ZNF500 mRNA expression was significantly higher in both paired and unpaired breast cancer samples than that in non-cancerous samples (Figure 1B and Additional file 3: Figure S1A). We evaluated ZNF500 mRNA expression among breast cancers within different subtypes; ZNF500 displayed the highest expression in luminal A type within the best prognosis and lowest expression in the basal-like type within the worst prognosis (Figure 1C). ZNF500 expression in non-triple-negative breast cancer and non-basal-like breast cancer patients was significantly higher than that in triple-negative breast cancer (TNBC) or basal-like breast cancer patients (Figure1D and Additional file 3: Figure S1B). Similarly, ZNF500 expression was significantly higher in estrogen receptor (ER)- or progesterone receptor (PR)-positive breast cancer patients than that in ER- or PR-negative breast cancer patients but visibly lower in HER2-positive breast cancer patients than that in HER2-absent patients (Additional file 3: Figure S1C-E). Moreover, ZNF500 expression was negatively correlated with advanced SBR grading(22) and high NPI scores (23)(Figures 1E-F). The receiver operating characteristic curve drawn based on ZNF500 levels demonstrated that ZNF500 might be an effective indicator of better prognosis (area under curve= 0.634, Additional file 3:Figure S1F). Kaplan–Meier analysis revealed that the overall survival time (OS) of patients with high ZNF500 expression was significantly longer than that of patients with negative expression (Figure 1G). Accordingly, the risk score curve also indicated that the survival time and survival rate of breast cancer patients with high ZNF500 expression was considerably higher than those of patients with low ZNF500 expression (Figure 1H). Univariate Cox regression analysis suggested that ZNF500 could be considered an independent risk prognostic factor for patients with breast cancer (Additional file 3: Figure S1G). ZNF500 expression in patients with wild-type (WT) p53 was significantly higher than that in patients with mutated p53 tissues at DNA, mRNA, and protein levels (Figure 1I and Additional file 3: Figure S1H-I).
Western blot analysis of 12 fresh breast cancer tissues and paired adjacent normal tissues revealed that ZNF500 protein levels were significantly higher in breast cancer tissues than those in non-cancerous tissues (P = 0.0089, Figure 1J and Additional file 3: Figure S1J). Subsequent immunohistochemical staining of 157 breast cancer samples and 61 normal breast tissue samples indicated that ZNF500 was highly expressed in the nucleus of breast cancer cells with dim cytosolic expression, and positive rate of ZNF500 (59.2%, 93/157) in breast cancer was significantly higher than that in normal breast tissue (10%, 6/61, P <0.01, Figure 1K-M). Statistical analysis indicated that ZNF500 expression was negatively correlated with p53 mutation (P = 0.001), TNBC (P = 0.0013), advanced TNM stage (P = 0.018), and lymph node metastasis (P = 0.014, Table 1). Kaplan–Meier analysis revealed that the OS of breast cancer patients with high ZNF500 expression (141.571 ± 3.203) was significantly longer than that of patients with negative ZNF500 expression (121.197 ± 5.847, P = 0.001, Figure 1N). Cox univariate and multivariate analyses revealed that, together with advanced TNM stage, ZNF500 expression could be considered an independent prognostic factor for breast cancer (P = 0.006, Table 2). We explored ZNF500 expression in diverse breast cancer cell lines and found that ZNF500 expression was higher in cells with p53-WT (MCF-7, ZR75-1, MDA-MB-361, and MDA-MB-175VII) than that in p53-mutant cells (SK-BR-3, MDA-MB-231, and BT549). However, ZNF500 showed increased expression in MDA-MB-453, a p53-null cell line (Figure 1O). Subsequent immunofluorescence (IF) assays indicated that ZNF500 was mainly localized in the nucleus of these cells (Figure 1P, Additional file 3: Figure S1K).
ZNF500 suppresses cell proliferation and induces cell cycle arrest in p53-WT breast cancer cells both in vitro and in vivo
Gene set enrichment analysis (GSEA) revealed that differentially expressed genes (DEGs) with low ZNF500 expression were closely enriched in the cell cycle process (Figure 2A and Additional file 3: Figure S2A). We overexpressed ZNF500 and knocked it out in several breast cancer cell lines with diverse p53 status (p53 WT cell lines: MCF-7, ZR-75-1, and MDA-MB-175VII; p53 mutant cell lines: SK-BR-3 and MDA-MB-231; and p53-null cell line: MDA-MB-453) using CRISPR-Cas9 guided by two different sgRNAs (Figure 2B and Additional file 3: Figure S3A-C). The MTT assay, colony formation assay, and EdU assay results indicated that proliferation in p53-WT cells was abrogated by overexpression or enhanced by the silencing of ZNF500 (Figure 2C-E and Additional file 3: Figure S3D-F); however, no significant changes were observed in the p53-mutant and -null cells (Additional file 3: Figure S3G-L). Therefore, p53-WT cells were selected for subsequent analysis.
We further explored the effects of ZNF500 expression on cell cycle progression using flow cytometry. As shown in Figure 2F and Additional file 3: Figure S4A, ZNF500 overexpression may induce cell cycle arrest in the G1 phase and shorten the S phase. Accordingly, the G1 phase was shortened, and the S phase was prolonged when ZNF500 was knocked out. Western blotting was performed to examine the key factors involved in the cell cycle. The results showed that ZNF500 overexpression or knock-out resulted in significantly downregulated or up-regulated CyclinD1 expression, respectively. Expression of the other cyclins did not show any changes (Figure 2G and Additional file 3: Figure S4B). We also performed a xenograft assay to explore the effect of ZNF500 expression on cell proliferation in vivo and found that the tumor volume was decreased or increased in ectopic or silenced ZNF500, respectively (Figure 2H).
ZNF500 prevents ubiquitination of p53 and activates the p53-p21-E2F4 signaling axis
We performed an RNA-array assay on 90 crucial target genes related to the cell cycle after overexpressing ZNF500. We found that the expression of two genes were upregulated (p21 and E2F4) and that of two genes were downregulated (CDK5R1 and MKI67) (Figure 3A and Additional file 3: Figure S5A). The GEPIA database was used to assess the correlation between ZNF500 and the target genes, which indicated that ZNF500 was positively correlated with p21 and E2F4 expression but not with CDK5R1 and MKI67 expression (Figure 3B and Additional file 3: Figure S5B). qPCR results confirmed that p21 and E2F4 mRNA expression was significantly elevated or decreased after overexpression or knockout of ZNF500, respectively (Figure 3C and Additional file 3: Figure S5C). Both p21 and E2F4 are classical downstream target genes of p53 signaling(24, 25). Therefore, we subsequently investigated whether the inhibition of the cell cycle induced by overexpression of ZNF500 is dependent on p53. Both p53 mRNA and protein levels were examined in ectopic or silenced ZNF500 cells, which indicated that p53 mRNA levels remained unaltered. The levels of p53 protein, phosphorylated p53 at Ser15, p21 and E2F4, were significantly enhanced or suppressed, whereas the expression of CDK4 was significantly decreased or increased after overexpressing or deleting ZNF500 (Figure 3D-E and Additional file 3: Figure S5D-E).
Then, we co-transfected ZNF500 overexpressing plasmid and p53 siRNA as well as the relative control, and the results indicated that up-regulation of p21 expression and down-regulation of CyclinD1 expression were counteracted by p53 knockdown (Figure 3F). Moreover, breast cancer cell proliferation was no longer suppressed (Figure 3G-H). The addition of CHX to block de novo protein synthesis showed that the degradation of p53 was significantly delayed after ZNF500 overexpression in MCF-7 and ZR-75-1 cells (Figure 3I). Subsequent WB and IF assays also revealed that overexpression of ZNF500 might prevent nuclear export, a process essential for p53 stability (Figure 3J-K). ZNF500 may be involved in the proteasome process, as indicated by the GSEA (Additional file 3: Figure S2A and S6A). We found that the elevation of p53 expression after ZNF500 overexpression was neutralized by the addition of MG132, a proteasome inhibitor (Additional file 3: Figure S6B). Furthermore, overexpression of ZNF500 significantly reduced the ubiquitination of p53 (Figure 3L).
ZNF500 directly binds to the C-terminal of p53 via its C2H2 domain
We performed a co-IP assay to examine the interaction between ZNF500 and p53. Our results indicated endogenous and exogenous interaction of ZNF500 with p53 (Figure 4A-B). GST pull-down assay revealed that ZNF500 may directly bind to p53 (Figure 4C). Subsequent IF assays revealed that both endogenous and exogenous p53 and ZNF500 were co-localized in the nucleus of breast cancer cells (Figure 4D-E). Then, we mapped the detailed domain responsible for the binding between ZNF500 and p53 and synthesized a series of splicing mutant plasmids for ZNF500 and p53 (Figure 4F-G). The co-IP assay results indicated that deletion of the C2H2 domain in ZNF500 and the C-terminal domain of p53 may abolish the interaction between ZNF500 and p53 (Figure 4H-I). The GST pull-down results also indicated that ZNF500 directly bound to the C-terminal domain of p53 (Figure 4J). We then overexpressed ZNF500-full length (FL), ZNF500-△C2H2, and NC plasmids in MCF-7 cells. The WB assay results revealed that overexpression of ZNF500-△C2H2 no longer up-regulated expression of p53 and its downstream factors, p21 and E2F4, compared to ZNF500-FL overexpression (Figure 4K). Ubiquitination of p53, as well as cell proliferation, were also not decreased or abrogated after overexpression of ZNF500-△C2H2 (Figure 4L-N). The xenograft assay also confirmed the effect of overexpression of ZNF500-△C2H2 in vivo (Figure 4O).
ZNF500 binds and stabilizes p53 in a manner that is competitive to MDM2
Our study revealed that ZNF500 binds and stabilizes p53 by preventing its ubiquitination, although ZNF500 is not an E3 ubiquitin ligase. Bioinformatics analysis revealed a negative correlation between ZNF500 expression and the IC50 of nutlin-3a, an MDM2-specific inhibitor (Additional file 3: Figure S7A). We knocked out ZNF500 and added nutlin-3a and found that suppression of p53 expression induced by silencing ZNF500 at least partially counteracted proliferation (Additional file 3: Figure S7B-C). We speculated that ZNF500 may stabilize p53 by modulating MDM2. A subsequent co-IP assay revealed that endogenous ZNF500, MDM2, and p53 could form a ternary complex (Figure 5A). However, the IF and WB assay results suggested that overexpression of ZNF500 did not affect the expression of MDM2 or its subcellular localization. (Figure 5B-C). Previous studies have indicated that most ubiquitin sites are localized in the C-terminal domain(26) , and MDM2 can also bind to the C-terminal of p53(27). We speculated whether ZNF500 may compete against MDM2 for the binding to p53, thus stabilizing it. We overexpressed p53 and MDM2 with increasing doses of ZNF500 in MCF-7 cells and performed a co-IP assay, which revealed that the binding between MDM2 and p53 was dose-dependently downregulated by increasing ZNF500 expression (Figure 5D). Similarly, the interaction between ZNF500 and p53 decreased in a dose-dependent manner when MDM2 was overexpressed (Figure 5E).
To further explore whether competitive binding to p53 between ZNF500 and MDM2 occurs under physiological conditions, a co-IP assay was performed between endogenous ZNF500 and p53 with increasing doses of MDM2. The results suggested that the endogenous interaction between ZNF500 and p53 was suppressed in a dose-dependent manner (Figure 5F). The endogenous interaction between MDM2 and p53 was also suppressed in a dose-dependent manner in ectopic ZNF500 (Figure 5G). Finally, we overexpressed ZNF500-FL, ZNF500-△C2H2, and the NC at different doses, and the co-IP assay results indicated that overexpression of ZNF500-△C2H2, rather than ZNF500-FL, could disrupt the interaction between MDM2 and p53 (Figure 5H).
ZNF500 accelerates DNA damage and sensitizes breast cancer cells to chemotherapy
Previous studies have demonstrated that p53 plays a crucial role in the process of DNA damage(28). We used camptothecin to induce DNA damage and found that the overexpression of ZNF500 may strengthen DNA damage, which was revealed by the up-regulation of γ-H2AX expression, the DNA damage maker, as well as its nuclear foci (Additional file 3: Figure S8A-B). Furthermore, compared with overexpression of ZNF500-FL, ectopic ZNF500-△C2H2 did not cause more DNA damage, as revealed by the expression and nuclear foci of γ-H2AX (Figure 6A-B). Bioinformatic analysis performed to explore the correlation between ZNF500 expression and chemotherapeutic drugs revealed that ZNF500 expression was significantly negatively correlated with resistance to doxorubicin and vinorelbine, two first-line neoadjuvant chemotherapy drugs for breast cancer patients (Figure 6C). Overexpression of ZNF500 visibly reduced the IC50 of doxorubicin and vinorelbine, accelerated DNA damage, and abrogated cell proliferation (Figure 6D-F).
Finally, we assessed IHC staining in specimens from 50 breast cancer patients with the p53-WT to evaluate whether the ZNF500-P53-E2F4 axis existed in human breast cancer specimens. The results suggested that ZNF500 was significantly positively correlated with p53 (P = 0.022, r = 0.342) and E2F4 (P = 0.004, r = 0.436) expression (Figure 6G, Table 3). We also assessed ZNF500 expression in specimens from patients with diverse therapeutic effects evaluated by Miller/Payne Grades after neoadjuvant chemotherapy. IHC staining results indicated that ZNF500 expression in sensitive patients (Miller/Payne Grade 1–2) was significantly lower than that in resistant patients (Miller/Payne Grade 3–5, P = 0.012, Figure 6H).