p53 downregulates PD-L1 expression via miR-34a to inhibit the growth of triple-negative breast cancer cells: a potential clinical immunotherapeutic target

Compared with other breast cancer subtypes, triple-negative breast cancer (TNBC) has poorer responses to therapy and lower overall survival rates. The use of an inhibitor of immune checkpoint programmed cell death ligand 1 (PD-L1) is a promising treatment strategy and is approved for malignant tumors, especially for TNBC. p53 regulates various biological processes, but the association between p53 and immune evasion remains unknown. miR-34a is a known tumor suppressor and p53-regulated miRNA that is downregulated in several cancers; however, it has not been reported in TNBC. Herein, we aimed to explore the regulatory signaling axis among p53, miR-34a and PD-L1 in TNBC cells in vivo and in tissue and to improve our understanding of immunotherapy for TNBC. p53-EGFP, p53-siRNA and miR-34a mimics were transfected into TNBC cell lines, and the interaction between miR-34a and PD-L1 was analyzed via dual-luciferase reporter assays. We found that p53 could inhibit the expression of PD-L1 via miR-34a and that miR-34a could inhibit both cell activity and migration and promoted apoptosis and cytotoxicity in TNBC. Furthermore, miR-34a agomir was injected into MDA-MB-231 tumors of nude mice. The results showed that miR-34a could inhibit tumor growth and downregulate the expression of PD-L1 in vivo. A total of 133 TNBC tissue samples were analyzed by immunochemistry; the proportion of positive expression of PD-L1 was 57.14% (76/133), and the proportion of samples with negative expression of PD-L1 was 42.86% (57/133). Our research may provide a novel potential target for TNBC.


Introduction
According to the new statistical data on tumor incidence rates released by the WHO in 2020 [1], breast cancer has surpassed lung cancer as the most commonly diagnosed cancer. As an important clinical subtype of breast cancer, TNBC has received increased amounts of attention due to its high degree of malignancy. Combined anthracyclines with cyclophosphamide and taxane-based regimens are the standard treatment for TNBC; unfortunately, they induce only an approximately 40% pathological response [2]. Thus, developing a new effective therapy is of utmost importance. Following the development of immune checkpoint targets, such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1), the treatment plans for TNBC are changing. PD-L1 is the main ligand of PD-1. PD-1 binds to PD-L1 to predominantly regulate effector T cell activity in tissues and tumors [3]. In light of the function of PD-L1 in immunotherapy, PD-L1 has received considerable amounts of attention from the research community. Mittendorf et al. revealed future research avenues in the immunotherapy of early-stage TNBC [4]. Moreover, an anti-PD-L1 regimen with atezolizumab and nab-paclitaxel has been approved for advanced TNBC expressing PD-L1 in clinical treatment [5]. However, the mechanisms of PD-L1-mediated treatment in TNBC have not yet been clarified.
As the guardian of the genome, TP53 has evolved to contribute to the regulation of almost every facet of cell behavior, including proliferation, growth, DNA repair, cell death, and cell survival [6]. Moreover, p53 could activate inflammation and the antitumor immune response. A study has shown that p53 is associated with immune checkpoint regulators [7]. In addition, p53 plays an important role in the inhibition of TNBC cells. Kollareddy et al. found that the expression of p53 could affect cell cycle distribution and severely perturb invasion in a TNBC cell line [8]. Yang et al. found that the acetylation of p53 could inhibit the growth of TNBC cells [9]. However, how p53 is involved in tumor immune evasion, especially in TNBC, is one of the unclear mechanisms with respect to immunotherapy.
MicroRNAs (miRNAs) compose a family of 21 ~ 25-nucleotide small RNAs that negatively regulate gene expression at the posttranscriptional level [10]. A modest number of miR-NAs, such as let-7, miR-34, and miR-200, lose their function in a very broad range of cancer types and play a central role in the regulation of tumor suppression [11]. Several p53-regulated miRNAs including the miR-17-92 cluster 127, miR-145 and let-7 have also been implicated in adaptive and innate immunity [12]. Previous studies have shown that miR-34a contains a consensus p53-binding site and that a reduction in miR-34 function attenuates p53-mediated cell death [13]. In another study, miR-34a altered the expression of PD-L1 in B-cell lymphomas [14]. Notably, studies of miR-34a in TNBC have rarely been reported.
Based on these results, using MDA-MB-231 cells and BT-549 cells, we found that p53 could regulate the expression of PD-L1 via miR-34a. miR-34a was demonstrated to significantly inhibit TNBC cells and tumor growth both in cells and in tumor-bearing mice. The expression of PD-L1 in paraffin sections of TNBC patients was analyzed and discussed. This finding, in which p53 was found to be associated with tumor immune evasion through this signaling axis, provided a novel potential target, improving our understanding of therapy for TNBC.

Dual-luciferase reporter assays
Binding sites between miR-34a-5p and PD-L1 were predicted via miRanda. Wild-type (WT) and mutant-type (MUT) plasmids were synthesized by GeneCopoeia. The plasmids and miR-34a-5p mimics or negative control (NC) mimics were cotransfected with TNBC cells by Lipofectamine 3000. A dual-luciferase reporter assay system (E1910, Promega) was used to perform the luciferase assays.

Cell counting kit-8 (CCK-8) assays
Cells transfected with miR-34a mimics were seeded into 96-well plates for 24 h, 36 h, and 48 h. 10 µL CCK-8 reagent was added to each well and incubated at 37 °C with 5% CO 2 . Varioskan LUX (Thermo Fisher) was used to measure the absorbance at 450 nm. The experiment was repeated three times, and the results were quantified.

Flow cytometry
Cells were transfected with miR-34a mimics, the cells were washed with PBS and trypsinized with 0.25% trypsin solution without EDTA. The cells were incubated with Annexin-V-FITC and PI and then analyzed by flow cytometry.

Calcein-AM/PI assays
Cells were transfected with miR-34a mimics, 250 µL of calcein-AM/PI reagent was added to each well and incubated at 37 °C with 5% CO 2 for 30 min in the dark, after which images were collected under an inverted light microscope equipped with a camera (Nikon). The experiment was repeated three times, and the results were quantified.

Wound healing assays
miR-34a mimics were transfected into cells and wounds were created by scratching the cells, and images were taken under a microscope. The cells were cultured in 2% FBS medium at 37 °C with 5% CO 2 for 24 h, and images were collected under an inverted light microscope equipped with a camera (Nikon). The experiment was repeated three times, and the results were quantified.

In vivo antitumor activities
Six-week-old female nude mice were injected subcutaneously with 1 × 10 7 MDA-MB-231 cells. When the tumors reached 5 mm × 5 mm in diameter, the mice were randomly divided into 2 groups, with 5 mice in each group. According to the manufacturer's recommendations and reference [15], one group was intratumorally injected with 5 nmol of miR-34a-5p agomir in 50 µL of PBS every 4 days, and the other group was intratumorally injected with 50 µL of PBS every 4 days. The tumor volume was calculated as tumor volume (mm 3 ) = [length (mm) × width (mm) 2 ] / 2. The animals were euthanized at the end of experiment.

Immunohistochemistry (IHC)
Mammary tissue specimens were collected from 133 patients with triple-negative breast cancer who were diagnosed at the Clinical Pathology Diagnosis Center of Chongqing Medical University between 2013 and 2017.
The sections were cut from paraffin blocks, deparaffinized with xylene and then rehydrated in different concentrations of ethanol. The slides were microwaved in citrate buffer for 10 min. The slides were treated with endogenous peroxidase inhibitor for 25 min at room temperature and incubated with rabbit monoclonal anti-PD-L1 antibody (#13684, 1:100, Cell Signaling Technology) overnight at 4 °C. A universal twostep test kit (pv9000, ZSGB-BIO) and a 3,3'-diaminobenzidine (DAB) staining kit (ZLI-9018, ZSGB-BIO) were used for immunohistochemistry following the manufacturer's instructions.

Evaluation of immunostaining
The staining results of the tumor epithelium were assessed by the intensity of staining and the proportion of positive cells. The staining intensity and positive proportion of PD-L1 were classified as 0, 1, 2, and 3 (staining intensity score: no staining score was 0, light yellow score was 1, brown score was 2, dark brown score was 3; positive proportion score: no positive cells were scored 0, the number of positive cells less than 25% was scored 1, the number of positive cells 25% ~ 50% was scored 2, and the number of positive cells more than 50% was scored 3). The total score was calculated as score = staining intensity score × positive proportion score. A total score ≥ 2 is positive, and a score < 2 is negative.

Statistical analysis
All experimental data were analyzed via IBM SPSS Statistics 22.0, and statistical drawings were constructed by GraphPad Prism 9.0. Student's t-test was used for comparisons among groups. The Kaplan-Meier method and log-rank test were employed for survival analysis. The chi-square test was used to analyze the relationship between PD-L1 expression and clinicopathological factors. P < 0.05 was considered statically significant.

p53 regulate miR-34a and PD-L1
To investigate the role of p53 in miR-34a and PD-L1 regulation, we regulated the expression of p53 in the TNBC cell lines. TNBC cells were transfected with the p53-EGFP plasmids or EGFP plasmids to overexpress p53. As shown in Fig. 1 A ~ B, WB demonstrated that cells transfected with p53-EGFP showed a notably higher p53 protein expression level and a significant decrease in PD-L1 protein level compared to those of the blank groups and EGFP groups in TNBC cell lines (P < 0.05; Fig. 1 A ~ B). Moreover, RT-qPCR demonstrated that miR-34a was upregulated substantially at the mRNA level compared to that of the blank groups and EGFP groups in both cell lines (P < 0.05; Fig. 1 C ~ D). Moreover, we transfected p53-siRNA and NC-siRNA into TNBC cell lines for 48 h to knock down p53. Compared with those in the blank group, NC-siRNA group and GAPDH-siRNA group, GAPDH protein levels in the GAPDH-siRNA group were prominently reduced, p53 protein levels in the p53-siRNA group were prominently reduced, and PD-L1 protein levels were markedly enhanced according to WB analysis (P < 0.05; Fig. 1E ~ F). RT-qPCR was also performed, which demonstrated that miR-34a was apparently downregulated, miR-34a decreased to 0.59, 0.66 and 0.52-fold in MDA-MB-231 cells, 0.72, 0.82 and 0.79fold in BT-549 cells (P < 0.05; Fig. 1G ~ H). Overall, we confirmed that p53 could upregulate miR-34a and downregulate PD-L1 in TNBC cells.

PD-L1 is a target of miR-34a
To demonstrate our hypothesis about the relationship between miR-34a and PD-L1, we transfected miR-34a mimics into TNBC cells and performed WB and RT-qPCR to detect and measure the expression of PD-L1 and miR-34a, respectively. As shown in Fig. 2A ~ B, the expression of miR-34a was markedly enhanced in the miR-34a mimic group (P < 0.05; Fig. 2A ~ B). Moreover, WB was performed, which showed that the protein expression levels of PD-L1 were significantly downregulated in the miR-34a mimic group (P < 0.05; Fig. 2 C ~ D). Therefore, we further investigated the relationship between miR-34a and PD-L1 in TNBC. Using the miRanda database, we found that there are potential binding sites between miR-34a-5p and the 3'-UTR of PD-L1, indicating that PD-L1 might be regulated by miR-34a-5p (Fig. 2E). To further confirm the target relationship between miR-34a and PD-L1, we constructed dual-luciferase plasmids and used a dual-luciferase reporter gene assay. Compared with that in the PD-L1 WT + mimics NC group, the luciferase activity in the PD-L1 WT + miR-34a mimic groups was inhibited but not in the PD-L1 MUT group (P < 0.05; Fig. 2 F ~ G). Based on the above experimental results, we conclude that PD-L1 is a target of miR-34a in TNBC.

miR-34a inhibited cell activity and migration, increased cytotoxicity, and promoted apoptosis in TNBC cells
A series of experiments were conducted to explore the effect of miR-34a on TNBC cells. miR-34a mimics were transfected into TNBC cells, and CCK-8 assays were performed. The results showed that miR-34a inhibited the activity of TNBC cells at 24 h, 36 h, and 48 h post-transfection ( Fig. 3 A ~ B). We further evaluated cell apoptosis by flow cytometry in these cells and revealed that miR-34a promoted apoptosis in TNBC cells (Fig. 3 C). Then, cell cytotoxicity was detected by calcein-AM/PI assays. While the control groups showed no marked cytotoxicity to TNBC cells, a host of dead cells was observed in the miR-34a transfection group (Fig. 3D ~ E). In addition, the results of the wound healing assay suggested that miR-34a inhibited TNBC cell migration (Fig. 3 F ~ G). Taken together, these data indicated that miR-34a inhibited cell activity and migration, increased cytotoxicity, and promoted apoptosis in TNBC cells.

miR-34a inhibited tumor growth in vivo
The effect of miR-34a on TNBC cells was verified, and we next investigated whether miR-34a might exert antitumor effects. To assess the therapeutic effect on MDA-MB-231 tumors in nude mice, we treated the mice with 5 nmol miR-34a-5p agomir or PBS starting when tumors grew to 5 mm × 5 mm in diameter and sacrificed all mice 24 h after the fifth treatment (Fig. 4 A). As shown in Fig. 4B, the nude mice were observed at 0 days and 16 days post-injection, and the results obviously showed that the miR-34a agomir could notably inhibit tumor growth in vivo, and the results were similar to those obtained in vitro. The mice weight and tumor volume were measured every 4 days (Fig. 4 C ~ D). There was no significant difference in mice weight but a decreased tumor volume at the termination of the experiment in the miR-34a agomir group compared with the control group. The miR-34a expression level was prominently enhanced in the tumor tissue obtained from the mice injected with miR-34a agomir compared with that of the mice injected with an equal volume of PBS at the end of the experiment (Fig. 4E). Importantly, the protein expression levels of PD-L1 in the two mice in the miR-34a groups were both lower than those in the control group (Fig. 4 F).

PD-L1 expression in TNBC tissues
The proportion of cases with positive expression of PD-L1 among 133 TNBC cases was 57.14% (76/133), and the proportion of cases with negative expression of PD-L1 among 133 TNBC cases was 42.86% (57/133) (Fig. 5 A). The associations between PD-L1 expression and clinicopathological factors in the studied cohort are summarized in Table S1. No significant difference was noted in terms of age (P = 0.960), tumor size (P = 0.191), histological grade (P = 0.082), lymphatic metastasis (P = 0.662) or Ki67 index (P = 0.420) between the PD-L1-positive group and PD-L1-negative group. Furthermore, Kaplan-Meier survival curve analysis based on the clinical data of 133 TNBC samples revealed no significant difference between the PD-L1-positive group and the PD-L1-negative group (P = 0.188) (Fig. 5B).

Discussion
Due to the lack of specific targets and no specific treatment plan, TNBC is called refractory breast cancer and has the characteristics of fast growth, strong invasion and is associated with poor therapeutic effect. Thus, developing a new effective therapy is necessary. Immunotherapy is currently commonly used in the treatment of TNBC. More recently, immune checkpoint inhibitors (CPIs) that target cytotoxic T lymphocyte-associated protein 4 and PD-1 or its ligand PD-L1 have shown robust clinical responses across various cancer types [16]. However, few studies have reported the mechanisms of PD-L1-mediated treatment in TNBC. In this study, we found that p53 could upregulate miR-34a and directly interact with PD-L1 through miR-34a to downregulate PD-L1 in TNBC (Fig. S1). Our study revealed a new signaling axis regulating PD-L1 in TNBC.
Previous studies have shown that p53 could activate inflammation and the antitumor immune response via direct transcriptional regulation of UL16-binding protein 1 (ULBP1) and ULBP2 [17][18][19]. However, the involvement of p53 in tumor immune evasion is poorly understood. In our study, the dual-luciferase reporter assay showed that miR-34a directly targeted the 3' UTR of PD-L1. Furthermore, we also found an association between p53 and tumor immune evasion by downregulating PD-L1 via miR-34a in cells in vivo and in tissue, further complementing recent findings implicating p53 and miR-34a in immune cell regulation, such as cellular senescence [13], apoptosis and gene expression [20]. Although the wild type and mutant type of p53 have not been discussed in this study, the whole p53 protein we detected has indeed obtained results, and we will further explore the mutation of p53 in the future. The association between p53 and PD-L1 has also been researched in other cancers, such as non-small cell lung cancer, the results of which are consistent with ours [21], whereas such a regulatory mechanism in TNBC was a novel discovery. These findings improve our understanding of immunotherapy for TNBC and provide a novel potential immunotherapeutic target for TNBC.
In this study, we verified the antitumor efficacy of miR-34a in TNBC both in vitro and in vivo. As the first miRNA genes that were found to be directly regulated by p53 [22], the miR-34 family participates in tumor suppression as part of the p53 network [13]. According to previous studies, deficiency or loss of miR-34a expression occurs frequently in multiple cancers, such as pancreatic cancer [23]. Therefore, we overexpressed miR-34a in TNBC cells, and the results suggested that upregulation of miR-34a could inhibit cell activity and migration, increase cytotoxicity, and promote apoptosis in TNBC cells. These results supported our hypothesis. Evidence to support this is also provided in acute myeloid leukemia [24]. Additionally, our data demonstrated that miR-34a could inhibit tumor growth effectively in vivo and downregulate the expression of PD-L1 in vivo. The efficacy of miR-34a complexation in prostate cancer and lymphoma mouse models was indicated to inhibit the growth of tumors [25,26], which is consistent with our data.
Based on previous studies, TNBC cells express PD-L1 more than other breast cancer subtypes [27], and PD-L1 is overexpressed in most TNBC patients [28]. We performed immunohistochemical staining on 133 TNBC tissues to detect the expression of PD-L1. The results showed that 57.1% of patients had positive PD-L1 expression and 42.9% of patients had negative PD-L1 expression. These results indicated that positive PD-L1 expression is more common in TNBC, which is consistent with previous studies. Although, compared with that of the PD-L1-negative group, the overall survival rate of the positive group had a decreasing trend, a limitation of this study is that we did not find a significant association between PD-L1 and the overall survival rate, and a smaller sample size and loss of clinical information may be important factors affecting these results. We will continue to explore this in future research.

Conclusion
Taken together, our results revealed that p53 is also associated with immune evasion, providing new insight into the relationship between miR-34a and PD-L1 expression in patients with TNBC and providing a potential possibility for immunotherapy of TNBC.

Conflict of interest
The authors have declared that no competing interest exists.
Ethical approval All the animal procedures were performed in accordance with the Guidelines of the Ministry of Science and Technology of Health Guide for Care and Use of Laboratory Animals, China, and approved by the Institutional Ethics Committee (IEC) of Chongqing Medical University. All the patients signed an informed consent form before surgery and agreed that their pathological specimens could be used for scientific research.