p66α Suppresses Breast Cancer Cell Growth and Migration by Acting as Co-Activator of p53

p66α is a GATA zinc finger domain-containing transcription factor that has been shown to be essential for gene silencing by participating in the NuRD complex. Several studies have suggested that p66α is a risk gene for a wide spectrum of diseases such as diabetes, schizophrenia, and breast cancer; however, its biological role has not been defined. Here, we report that p66α functions as a tumor suppressor to inhibit breast cancer cell growth and migration, evidenced by the fact that the depletion of p66α results in accelerated tumor growth and migration of breast cancer cells. Mechanistically, immunoprecipitation assays identify p66α as a p53-interacting protein that binds the DNA-binding domain of p53 molecule predominantly via its CR2 domain. Depletion of p66α in multiple breast cells results in decreased expression of p53 target genes, while over-expression of p66α results in increased expression of these target genes. Moreover, p66α promotes the transactivity of p53 by enhancing p53 binding at target promoters. Together, these findings demonstrate that p66α is a tumor suppressor by functioning as a co-activator of p53.

For transient transfection, plasmids were transiently transfected into cells at approximately 70% confluence using PEI reagent (#23966-2, polysciences, Warrington, PA, USA) according to the manufacturer's instructions. After 48 h of transfection, cells were harvested.
The stable cells were established via the viral infection method. For lentivirus packages, indicated plasmids were transfected into HEK-293T cells according to the transient transfection method. The supernatants containing viruses were collected at 48 h and 72 h after transfection and used to infect target cells. Twenty-four hours post-infection, puromycin (1 µg/mL for MDA-MB-231 and MCF-10A cells, 2 µg/mL for MCF-7 cells) was added to select the stable cells for 5 days.
GST-p53 and its truncation mutant proteins were expressed in E. coli BL21 and purified by using GST beads (17-

Reverse Transcription PCR (RT-PCR) and Quantitative Real-Time PCR (qRT-PCR)
Total RNA from stable cells was extracted using the TRIzol reagent (15596026, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription procedure was described previously; three micrograms of the treated total RNA were used for cDNA synthesis in a 20 µL reaction using SuperScript II reverse transcriptase (18064022, Invitrogen, Carlsbad, CA, USA) [48][49][50].
qRT-PCR was performed using Hieff SYBR Green Master Mix (11201ES03, Yeasen Biotech, Shanghai, China), and reactions were performed on Roche LightCycler ® 480 II Real-Time PCR System. Data were acquired and analyzed using the 2 −∆∆CT method with ACTIN as an endogenous control. Probes and primers used in qRT-PCR are listed in Table S1 (Supplementary Material).

Chromatin Immunoprecipitation (ChIP)
ChIP assays were carried out in MDA-MB-231-Luc shVector and shp66α cells. The cells were grown in 100 mm plates to 90% confluence and fixed by 1% formaldehyde for 10 min in the cell culture incubator. The cross-linking reaction was stopped by 0.125 M glycine in 1 × PBS at room temperature for 5 min. Cells were harvested, washed, and lysed in the ChIP lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5% vol/vol), TritonX-100 (1.0% vol/vol). The chromatin was sonicated to fragments ranging from 300-800 bp in size. Immunoprecipitation was performed using the antibody against p53 (mouse, DO-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse normal IgG (sc-2025, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoprecipitated DNAs were amplified by qRT-PCR. Primers used for ChIP qRT-PCR were listed in Table S2 (Supplementary Material).

Luciferase Reporter Assay
Luciferase reporter assay was performed in HEK-293T cells. After 24 h transfection, cells were harvested in luciferase lysis buffer. Firefly luciferase activity (MA0519, Meilunbio, Dalian, China) and β-galactosidase activity (631712, Takara, Tokyo, Japan) were measured using the Commercial reagents according to the manufacturer's instructions.

Cell Counting Assay
Cells (3000 per well) were plated in a 96-well plate containing 100 µL/well medium, and cell counting assays were performed according to the manufacturer's instructions of Cell Counting Kit-8 (CK04, Dojindo, Shanghai, China). Absorbance at 450 nm was quantified at different time points using the Multiskan MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The cell growth curve was drawn with the Graphpad Prism.

Cell Migration Assay
Migration assays were performed as described previously [50]. After being starved for about 12 h, stable cells were harvested using trypsin and counted. A total of 5 × 10 4 cells were resuspended in the serum-free medium and placed in the upper 8 µm pore transwell filters (3495, Corning Incorporated, Corning, NY, USA ). Complete DMEM medium was added to the bottom chamber as attractants. The chamber was incubated at 37 • C and in a 5% CO 2 atmosphere for 18 hours. The cells were fixed with 4% poly-formaldehyde for 15 min, and cells were stained with Coomassie Brilliant Blue (G250) for 30 min. Nonmigrated cells on the upper side of the filter were removed with a cotton swab. The chamber was washed with PBS 2-3 times and dried up at 37 • C [50]. The migrated cells on the bottom of filter were quantified by counting nine randomly chosen fields (20×) using microscopy in each well. Experiments were repeated in triplicates. The eluted proteins were resolved on 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for Western blotting and silver and colloidal staining analyses. The proteins located between 40-kDa and 55-kDa were excised from the gels and identified by standard mass spectrometry.

Subcutaneous Xenograft Assay
Female BALB/c nude mice were purchased from SLAC Laboratory Co. Ltd. (Shanghai, China) and divided into two groups randomly. The total number of 2 × 10 6 (in 100 µL of PBS per mouse) luciferase-expressing MDA-MB-231 cells with or without p66α depletion cells was injected into 6-week-old nude mice subcutaneously per group (n = 10). Three weeks after inoculation, the mice were euthanized and the tumors were dissected and fixed in 4% paraformaldehyde. The volume of tumors was estimated according to the formula V = 0.52 × L × W 2 .

Lung Metastasis Assay and Hematoxylin and Eosin (H&E) Staining
Female BALB/c nude mice were purchased from SLAC Laboratory Co. Ltd. (Shanghai, China) and divided into two groups randomly. To analyze lung metastasis, a total number of 2 × 10 6 (in 100 µL of PBS per mouse) luciferase-expressing MDA-MB-231 cells with or without p66α depletion cells were injected into the tail vein of 6-week-old female mice (n = 10 for each group). After inoculation, bioluminescence imaging of metastatic tumors was carried out using Xenogen IVIS Imaging System every two weeks.

Survival Analysis and Correlation Analysis
The survival analyses of p66α and p53 were generated from Kaplan-Meier plots online tools, which are capable of assessing the effect of 54 k genes (mRNA, miRNA, protein) on survival in 21 cancer types including breast cancer. The patient samples are split into two groups according to the expression level of p66α or p53. The two patient cohorts are compared by the Kaplan-Meier survival plot, and the hazard ratio with 95% confidence intervals and log-rank p value is calculated. (http://kmplot.com, accessed on 3 December 2021) The correlation analysis of p66α and p53 in breast cancer was generated with the online tools based on the TCGA database GEPIA (http://gepia.cancer-pku.cn/, accessed on 3 December 2021).

Statistical Analysis
Data presented as mean ± SD were analyzed by the independent Student's t test.

p66α Inhibits Breast Cancer Cell Growth
To examine if p66α participates in breast-cancer-relevant biological processes, we first performed extensive bioinformatics analyses to determine if there is a correlation between p66α expression level and the clinical outcome in breast cancer patients. We found that patients with the high level of p66α expression showed a better overall survival outcome ( Figure 1A). This observation prompted us to examine the effect of p66α on breast cancer cell growth. First, p66α was depleted in MCF-7 cells by using specific targeting shRNAs ( Figure 1B), and the cell growth rate was examined by CCK8 assays. Notably, depletion of p66α increased the growth of MCF-7 cells significantly ( Figure 1C). Conversely, ectopic expression of p66α inhibited the growth of MCF-7 cells ( Figure 1D,E). Similarly, knockdown of p66α in MDA-MB-231 resulted in increased cell growth ( Figure 1F,G), while overexpression of p66α significantly inhibited cell growth ( Figure 1H,I). Similar results were also observed in MCF-10A, which is classified as a normal breast cell line ( Figure  S1A,B). Next, we subcutaneously injected the MDA-MB-231-Luc-shp66α or MDA-MB-231-Luc-shVector cells into nude mice, which were euthanized to dissect the tumors 18 days post-injection. The tumor sizes were measured and showed that the volume of the tumors in the knockdown p66α group was remarkably larger than those in the control group ( Figure 1J,K), indicating that depletion of p66α enhances the tumor cell growth.

p66α Suppresses Breast Cancer Cell Migration and Metastasis
To determine the role of p66α in breast cancer cell migration, cells stably expressing shp66α or vector were subjected to transwell assays. Depletion of p66α expression markedly promoted cell migration in MCF-7 and MDA-MB-231 cells, respectively (Figure 2A Figure 2I). The luciferase signal intensities of lung metastatic nodules were significantly increased in the shp66α group compared with those in the control group ( Figure 2J), and H&E staining assays revealed that the metastatic foci in the shp66α group were dramatically increased in tissue sections of the lungs ( Figure 2K,L). Taken together, these observations demonstrate that p66α suppresses the migration and metastasis of breast cancer cells.

p66α Interacts with p53
To investigate how p66α elicits the suppressive effects on cell growth and migration, we performed an immunoprecipitation coupled mass spectrometry assay to explore p66αinteracting proteins. We established HEK-293T cells stably expressing Flag-p66α and performed affinity purification with Flag antibody. p53 was identified as a potential p66αinteracting protein. To confirm the interaction between p66α and p53, we first transiently co-expressed full-length p66α and p53 in HEK-293T cells, and co-immunoprecipitations were performed. Indeed, p66α robustly and reciprocally interacted with p53 in HEK-293T cells ( Figure 3A,B). To further confirm that a p66α-p53 interaction occurs within the endogenous proteins, whole-cell lysates prepared from HEK-293T-Flag-p66α cells were used for immunoprecipitation with antibody against p66α, and Western blotting assays showed that p66α could co-elute with endogenous p53 protein ( Figure 3C). Furthermore, indirect immunofluorescence staining showed that the two proteins co-existed in the nucleoplasm ( Figure 3D). Collectively, these data suggest that p53 is a p66α-interacting protein.

p66α Suppresses Breast Cancer Cell Migration and Metastasis
To determine the role of p66α in breast cancer cell migration, cells stably expressing shp66α or vector were subjected to transwell assays. Depletion of p66α expression markedly promoted cell migration in MCF-7 and MDA-MB-231 cells, respectively (Figure

The CR2 Domain of p66α and the DNA-Binding Domain of p53 Are Essential for the Interaction
To identify regions in p66α that are critical for the interaction between p66α and p53, p66α full-length or truncations were transiently co-expressed with GFP-p53 in HEK-293T cells. Co-IP assays demonstrated that CR1 only weakly bound p53, while the CR2 region robustly bound p53 ( Figure 3E,F).
To determine the domains in p53 protein required for interacting with p66α, the full length of GST-p53 and three truncated p53 proteins were purified and incubated with HA-p66α protein prepared from HEK-293T cells. The co-precipitation experiments were performed by using GST beads, and the co-eluted HA-p66α protein was examined by Western blot assays by using HA antibody. The truncation mutant consists of 100-291aa containing DNA-binding domain motif was capable of binding p66α, which was comparable to that of the full-length p66α, while the mutants comprising 1 -99aa or 292-393aa of p53 failed to bind p66α ( Figure 3G,H). Taken together, the CR2 domain of p66α and the DNA-binding domain (100 -291aa) of p53 are essential for the interaction.

p66α Promotes the Expression of p53 Target Genes
To determine if the p66α-p53 interaction impacts p53 to regulate its target genes, we examined the expression of the known p53 target genes that control cell growth such as BAX, GADD45A, NOXA, and that control cell migration such as PAI-1 in the MCF-7-shp66α or vector control cells by qRT-PCR. Notably, depletion of p66α decreased the expression of p53 target genes in MCF-7 cells ( Figure 4A,B). Moreover, the increase in p66α levels in MCF-7 cells promoted the expression of these targets ( Figure 4C,D), which is dependent on the expression of p53 ( Figure 4E,F). A similar effect of p66α levels on the expression of the p53 target genes was observed in MDA-MB-231 cells ( Figure 4G-L).

p66α Promotes the Transactivity of p53 by Enhancing p53 Binding at Target Promoters
To verify the effects of p66α on the transactivation ability of p53WT and p53R280K, we performed luciferase reporter assays with BAX-Luc or NOXA-Luc, which contained p53 responsive elements, respectively. Notably, both p53WT and p53R280K can transactivate BAX or NOXA, and a combination with p66α markedly increased BAXor NOXA-driven reporter activities ( Figure 5A,B).
To examine whether p66α affects the expression of p53, we detected the expression level of p53 in stable cells with knockdown or overexpression of p66α. The results showed that p66α did not affect the mRNA level of p53 ( Figure S2A-C), nor did it affect the protein level of p53 ( Figure S2D-F).
To determine the effects of p66α on the DNA-binding activity of p53, we performed ChIP assays in MDA-MB-231-shVector and MDA-MB-231-shp66α cells using antibodies specific to p53, and the enriched target DNA fragments were amplified by qPCR using primer sets flanking the functional p53 binding sites within target promoters. Depletion of p66α significantly decreased the binding of p53 to target promoters including BAX, NOXA, GADD45A, and PAI-1 ( Figure 5C). Taken together, these results indicate that p66α promotes the transactivity of p53 by enhancing p53 binding at target promoters.   The CR2 domain contains a GATA-type zinc finger motif. (F) p53 mainly interacted with the CR2 domain of p66α, but there was also a weak interaction between p53 and CR1 domain. The asterisk "*" means nonspecific bands. (G) Diagrams of full-length p53 and p53 deletion mutants. (H) The region between 100 and 291 amino acid residues of p53 bound p66α. In vitro translated p66α protein was subjected to GST pulldown assays using the purified GST fusion p53 proteins.

High Expression of p66α and p53 Is Positively Correlated and Predicts Good Prognosis in Breast Cancer
To determine the clinical relevance of p66α and p53, we analyzed a Kaplan-Meier plotter database and found that high expression of p53 also predicted good prognoses in patients with breast tumors ( Figure 6A). Moreover, the expression of p66α and p53 have a significant correlation in breast cancer samples ( Figure 6B).

High Expression of p66α and p53 Is Positively Correlated and Predicts Good Prognosis in Breast Cancer
To determine the clinical relevance of p66α and p53, we analyzed a Kaplan-Meier plotter database and found that high expression of p53 also predicted good prognoses in patients with breast tumors ( Figure 6A). Moreover, the expression of p66α and p53 have a significant correlation in breast cancer samples ( Figure 6B). ells 2021, 10, x FOR PEER REVIEW Figure 6. High expression of p66α and p53 is positively correlated with and predicts a good prognosis in brea Kaplan-Meier plot of the relapse-free survival of patients with breast cancer in whole data sets stratified by p53 e Scatter plot showed that p66α and p53 were positively correlated in breast cancer. The p value was calculated ranking correlation coefficient analysis. BRCA: breast invasive carcinoma (TCGA database). TPM: Transcripts P exon per Million mapped reads. (C) Schematic model for p66α mediated p53 transactivating genes expression. p6 with p53 and promoted the transcriptional activation ability of p53 by enhancing p53 binding in target promot genes that control cell growth, such as BAX, GADD45A, and NOXA, and that control cell migration such as PAI-1.

p66α Functions as a Tumor Suppressor of Breast Cancer Cells
Breast cancer is one of the most common cancers in women, which ha to-incidence ratio of 15% and alone accounts for 30% of female cancers [51 genous malignancy, it could be divided into three categories according to tures, including hormone-positive subtype expressing estrogen receptor (E terone receptor (PR+), HER2-positive subtype expressing human epiderm (HER2+), and triple-negative breast cancer (TNBC) (ER-, PR-, HER2-) [5 treatment is the most challenging within breast cancer for its higher aggr and lack of molecular targets [53][54][55], although there are a large number pressor genes encoding proteins that inhibit cell transformation in breast c TP53, Rb1, PTEN, BRCA1/2, CDKN1B, CDKN2A, etc.

p66α Functions as a Tumor Suppressor of Breast Cancer Cells
Breast cancer is one of the most common cancers in women, which has a mortality-toincidence ratio of 15% and alone accounts for 30% of female cancers [51]. As a heterogenous malignancy, it could be divided into three categories according to molecular features, including hormone-positive subtype expressing estrogen receptor (ER+) or progesterone receptor (PR+), HER2-positive subtype expressing human epidermal receptor 2 (HER2+), and triple-negative breast cancer (TNBC) (ER-, PR-, HER2-) [52]. The TNBC treatment is the most challenging within breast cancer for its higher aggressive feature and lack of molecular targets [53][54][55], although there are a large number of tumor suppressor genes encoding proteins that inhibit cell transformation in breast cancer, such as TP53, Rb1, PTEN, BRCA1/2, CDKN1B, CDKN2A, etc.
In this study, we demonstrate that p66α functions as a tumor suppressor that can inhibit breast cancer cell growth and migration. Expression of p66α can inhibit growth and migration of breast cancer cells MCF-7 and MDA-MB-231, as well as normal breast cells MCF-10A. Mechanistically, p66α functions as a novel co-activator of p53 to induce its target gene expression, and p66α promotes the transactivity of p53 by enhancing p53 binding at target promoters such as BAX, NOXA, GADD45A, and PAI-1, resulting in inhibition of cell growth and migration. Moreover, breast cancer patients having a higher level of p66α exhibit better prognosis. Taken together, our findings suggest that p66α could be a target for new strategies in the treatment of breast cancers.

p66α Functions as a Potent Co-Activator of p53 by Enhancing Its DNA-Binding Activity
Although p66α harbors a GATA motif within its CR2 region, there is no evidence to show that p66α can directly bind DNA sequences; rather, p66α acts like an adapter or a scaffold to participate in assembly of various protein complexes. Thus, the molecular mechanism by which p66α enhances the DNA-binding and transcriptional activity of p53 remains elusive and definitely needs to be explored further. For example, identifying the protein partners that associate with p53/p66α complex will provide new clues.
Another interesting observation is that p66α can enhance p53 target gene expression in MDA-MB-231 cells in a p53-dependent manner, since the p53 protein in MDA-MB-231 cells harbor the R280K mutation. R280K resides in the DNA-binding domain and results in impaired DNA-binding activity of p53 [56,57]. We found the p53R280K is also enriched at the promoters of target genes including BAX, NOXA, GADD45A, and PAI-1 in MDA-MB-231 cells. Moreover, the luciferase reporter assays on promoters of BAX and NOXA showed that both p53 WT and R280K alone can induce their promoter activities, and adding p66α can further induce these promoter-luc activities.
We reasoned that this may be explained by at least two possible mechanisms: (1) p53R280K mutant still retains DNA-binding activity. Malcikova reported that p53R280K lost most of its binding ability to the CDKN1A and retained about 35% binding ability to the BAX gene [58]. In another study, the DNA-binding ability of p53R280K to the GADD45A gene was reduced, but not completely lost [59]. (2) MDA-MB-231 cells express a relatively higher level of p66α protein compared to MCF-7 and MCF-10A cells. Our data show that p66α can enhance the DNA-binding activity of p53 to its target promoters. The binding activities of p53R280K to its target promoters in MDA-MB-231 cells may be attributed to p66α, which may overcome the effect of the mutation.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10123593/s1, Figure S1: p66α suppresses cell growth and migration in MCF-10A cell line, Figure S2: The protein and mRNA level of p53 is not affected by p66α expression or knockdown, Table S1: A list of primer sequences applied in qRT-PCR, Table S2