Apigenin suppresses the stem cell-like properties of triple-negative breast cancer cells by inhibiting YAP/TAZ activity

Triple-negative breast cancer (TNBC) remains a clinical challenge because of the absence of effective therapeutic targets. In TNBC, overexpression of YAP and TAZ correlates with bioactivities of cancer stem cells (CSCs), high histological grade, resistance to chemotherapy, and metastasis. Thus, YAP/TAZ may serve as potential therapeutic targets in TNBC. To identify YAP/TAZ inhibitors, in previous experiments, we screened a library of natural compounds by using YAP/TAZ luciferase reporter assay and identified apigenin as a potential inhibitor. In this study, we demonstrated that apigenin significantly suppressed the proliferation and migration of TNBC cells. Furthermore, we demonstrated that apigenin inhibited stemness features of TNBC cells in both in vitro and in vivo assays. Our mechanism study demonstrated that apigenin decreased YAP/TAZ activity and the expression of target genes, such as CTGF and CYR61, in TNBC cells. We also showed that apigenin disrupted the YAP/TAZ-TEADs protein–protein interaction and decreased expression of TAZ sensitized TNBC cells to apigenin treatment. Collectively, our studies suggest that apigenin is a promising therapeutic agent for the treatment of TNBC patients with high YAP/TAZ activity.


Introduction
Breast cancer is the most frequently diagnosed cancer in women worldwide 1,2 . It is highly heterogeneous and clinically classified into different subtypes based on the status of receptors 3 . Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes and constitutes around 15-20% of breast cancer cases 4 . Because of the lack of therapeutic targets, patients diagnosed with this type of breast cancer have a poor prognosis, along with a high rate of recurrence after chemotherapy 5,6 . Hence, the development of effective treatments for TNBC is an important unmet medical need.
Natural products and their derivatives have been used as anticancer agents and present a potential source of new drugs to combat cancer [22][23][24] . To identify potential YAP/ TAZ inhibitors, we previously performed a YAP/TAZ luciferase reporter activity-based screening of a library of natural products (Selleckchem, Houston, TX, USA), and found that apigenin decreased YAP/TAZ activity. In this study, we demonstrated that apigenin significantly suppressed the migration and CSCs properties of TNBC cells. We found that apigenin acted at least partially by inhibiting the YAP/TAZ-TEADs complex activity. These results confirmed that apigenin was a promising agent for the treatment of TNBC patients with high YAP/TAZ activity.

Apigenin inhibited cell viability in TNBC cells
We used the SRB protein assay to investigate the effect of apigenin on the proliferation of TNBC cells and found that apigenin inhibited cell proliferation in a dose-and time-dependent manner (Fig. 1b, c). The IC50 values of apigenin in MDA-MB-231 and MDA-MB-436 cells were approximately 33 and 30 μM, respectively, at 72 h. As shown in Supplementary Fig. 1, apigenin was relatively nontoxic to normal human breast cells. We also examined whether apigenin might affect the ability of TNBC cells to form colonies and found that apigenin indeed significantly reduced the rate of colony formation in both TNBC cell lines (Fig. 1d, e).

Apigenin attenuated migration in TNBC cells
We used wound-healing assays to evaluate the effects of apigenin on the migration of TNBC cells. Our results demonstrated that apigenin reduced gap closure at concentrations of 10 and 20 µM (Fig. 2a, b). We also conducted transwell migration assays on TNBC cells to confirm the above finding. As shown in Fig. 2c, apigenin treatment decreased the number of migrated cells in a dose-dependent manner. Statistical analysis indicated that apigenin caused a significant decrease in the migration of MDA-MB-231 and MDA-MB-436 cells (Fig. 2d).
Apigenin suppressed the stem cell-like properties and tumorigenic potential of TNBC cells We examined the anti-CSCs effects of apigenin over a range of concentrations that did not cause massive cell death (0-20 µM). We first investigated whether apigenin could decrease the percentage of the CD44 + /CD24 − subpopulation in TNBC cells. As shown in Fig. 3a, b, apigenin treatment decreased the CD44 + /CD24 − CSC subpopulations in MDA-MB-231 and MDA-MB-436 cells. Consistent with these findings, we observed that apigenin significantly decreased the number of mammospheres formed from both cell lines, indicating a reduction in self-renewal capability (Fig. 3c, d). These results indicated that apigenin decreased CSC-like traits on TNBC cells in vitro.
We also examined the effects of apigenin on the tumorinitiating properties of TNBC cells using an in vivo limited dilution assay. As shown in Fig. 4a, 1 × 10 6 and 1 × 10 5 MDA-MB-231 cells formed tumor xenografts with 100% efficiency in the control group, but the tumor formation efficiency of the apigenin-treated group decreased to 50% and 20%, respectively. When cells were seeded at a density of 1 × 10 4 cells per site, the tumor formation efficiency in the apigenin-treated group decreased to 0%, whereas the control group retained 75% of its efficiency. Additionally, the onset of tumor growth in the apigenin-treated group was delayed compared with the control, DMSO-treated group (Fig. 4b). Apigenin treatment also significantly decreased tumor volumes and weights ( Fig. 4c-e).

Apigenin reversed the malignant phenotype of TNBC cells by inhibiting YAP/TAZ transcription activities
To explore whether apigenin inhibited YAP/TAZ transcriptional activity in TNBC cells, we used the luciferase reporter assay. Our results demonstrated that apigenin significantly decreased YAP/TAZ activity in TNBC cells (Fig. 5a). To further confirm that apigenin inhibited YAP/TAZ transcriptional activity, we evaluated the expression of the YAP/TAZ target genes, CTGF and CYR61, and found that apigenin significantly decreased the mRNA and protein levels of CTGF and CYR61 in a dose-dependent manner ( Fig. 5b-d). The protein levels of YAP and TAZ, however, were not affected by apigenin treatment.

Apigenin disrupted YAP/TAZ-TEAD interaction in TNBC cells
We previously verified that the TAZ-TEADs interaction was indispensable for TAZ in maintaining CSCs traits of breast cancer cells 25 . Therefore, we hypothesized that apigenin could act by disrupting the interaction of TAZ and TEADs in TNBC cells, and we performed coimmunoprecipitation assays to explore the interaction between TAZ and TEADs. Apigenin disrupted the interaction of TAZ and TEADs in MDA-MB-231 and MDA-MB-436 cells (Fig. 6a, b), as well as the interaction of YAP and TEADs in MDA-MB-436 cells. To further explore whether TAZ played a major role in the anticancer activities of apigenin, we decreased TAZ expression in TNBC cells by CRISPR/Cas9-mediated genome editing. As expected, knockdown of TAZ sensitized TNBC cells to apigenin treatment, with a 50-70% decrease in IC50 (Fig. 6c, d). These results suggested that the reversion of the malignant phenotype of TNBC cells by apigenin was, at least in part, due to inhibition of the YAP/TAZ-TEADs complex activity.

Discussion
The theory of CSCs proposed that a small population of cancer cells had features similar to normal stem cells and that these cells played important roles in tumor initiation and maintenance 26 . In recent studies, CSCs have been isolated and identified in several solid tumors, including breast cancer 27 . In TNBC, breast CSCs were highly enriched and related to chemotherapy resistance, tumor relapse, and metastasis [28][29][30] . Therefore, the development of therapy targeting CSCs might benefit patients with TNBC.
Apigenin is a widely distributed flavonoid in vegetables and fruits. Recent studies have reported that apigenin has anticancer activity [31][32][33] , but the effects and underlying mechanisms of apigenin on TNBC have remained largely unexplored. In our drug-screening study, we found that apigenin decreased YAP/TAZ luciferase reporter activity. Therefore, we hypothesized that apigenin inhibited the migration and properties of CSCs in TNBC cells by regulating the Hippo-YAP/TAZ signaling pathway.
The population of CD44 + /CD24 − cells was recognized as CSCs in breast cancer 34 . In the current study, we used MDA-MB-231 and MDA-MB-436 cells to investigate the functions and mechanisms of apigenin on TNBC cells, because both cell lines overexpressed TAZ and possessed a high number of CD44 + /CD24 − cells. Our results showed that apigenin significantly suppressed proliferation and migration of TNBC cells. We further demonstrated that apigenin robustly inhibited features of stemness in TNBC cells, evidenced by a decrease in the CD44 + /CD24 − CSC subpopulation and mammosphere formation. Also, limiting dilution analysis of tumorigenesis confirmed that apigenin inhibited the tumor-initiating properties of TNBC in vivo.
Next, we investigated the molecular mechanisms underlying the anticancer effects of apigenin. We verified that apigenin inhibited YAP/TAZ transcriptional activity in TNBC cells, as assayed by luciferase reporter assays. Consistently, apigenin significantly decreased the expression of CTGF and CYR61, two YAP/TAZ-regulated genes, at both the mRNA and protein level.
YAP and TAZ mainly interact with TEADs to induce the expression of downstream genes that are involved in cell proliferation and migration [35][36][37] . Therefore, the YAP/ TAZ-TEADs complex may serve as a potential therapeutic target. In the current study, we investigated the effect of apigenin on the interaction of YAP/TAZ and TEADs. Our results demonstrated that apigenin disrupted the YAP/TAZ-TEADs protein-protein interaction in MDA-MB-436 cells. In MDA-MB-231 cells, we found that apigenin disrupted the TAZ-TEADs interaction. We did not find evidence for interaction between YAP and TEADs in MDA-MB-231 cells. We assume that TAZ, but not YAP, plays a key role in the downstream effects of the Hippo pathway in MDA-MB-231 cells. Furthermore, CRISPR/Cas9-mediated knockout of TAZ sensitized TNBC cells to apigenin treatment.
In conclusion, we demonstrated for the first time that apigenin inhibited YAP/TAZ activity in TNBC cells. The effects of apigenin on TNBC cells are mediated, at least in part, by disrupting the YAP/TAZ-TEADs protein-protein interaction, which is depicted in the model shown in Fig. 6e. These results suggested that apigenin might offer a novel therapeutic option for TNBC patients with high YAP/TAZ activity.
Cell proliferation and colony formation assay SRB protein assay was used in this study to analyze cell proliferation 40 . Briefly, TNBC cells were plated in 96well plates (6 × 10 3 cells per well) and grown overnight. Apigenin (0-64 μM) was added to the indicated wells and plates were incubated for 24, 48, or 72 h. The IC50 value of apigenin in TNBC cells was calculated using GraphPad Software. For colony formation, MDA-MB-231 and MDA-MB-436 cells (1000 cells per well) were seeded on 24-well plates. The experimental groups were treated with the indicated concentrations of apigenin for 72 h and then replaced for fresh medium. The plates were finally stained with 0.2% crystal violet (Amresco#0528) and counted. Mammosphere formation assay TNBC cells were treated with different concentrations of apigenin for 72 h and mammosphere formation assay was conducted as previously decribed 25 . In brief, apigenin-treated and DMSO-treated cells were plated into ultra-low attachment plates at the density of 10,000 cells per well (Corning, NY, USA). On day 10, images were taken at 4x magnification and counted. Wound-healing and transwell migration assays TNBC cells were seeded onto 6-well plates to reach 90% confluence. A sterile P200 pipette tip was used to create a scratch in the middle of each well. Cell debris was removed, and the remaining cells were maintained in the absence or presence of apigenin for 24 h. Migrated cells were photographed at 0, 12, or 24 h by using a Zeiss inverted microscope (Zeiss GmbH, Germany; magnification, 4 × ). For transwell migration assay, TNBC cells were pretreated with the indicated concentrations of apigenin for 24 h. Cells were resuspended in serum-free medium, plated into transwell chambers (8-µm pore size; Corning), and cultured for 24 h. Migrated cells were stained with crystal violet and counted under the microscope.

Luciferase reporter assay
Luciferase reporter assay was performed using previously described methods 41 . Briefly, MDA-MB-231 or MDA-MB-436 cells were plated in 96-well plates and grown overnight. Cells were co-transfected with 0.2 μg of 8xGTIIC-luciferase reporter plasmid and 10 ng of pRL-CMV plasmid using Lipofectamine 3000 reagent. Indicated concentrations of apigenin (5, 10, and 20 µM) were added to the cells at 24 h post-transfection. Luciferase activity was detected with the DLR™ assay system (Promega#E1910), according to the manufacturers' protocol.

Real-time PCR
TRIzol reagent (Thermo, USA) was applied for total RNA extraction. The RNA was reverse transcribed using the cDNA Synthesis Kit (Thermo#K1622) following the The cells were harvested, and the effect of apigenin on the YAP/TAZ and TEADs interaction was analyzed by immunoprecipitation assay. c, d TAZ knockout sensitized TNBC cells to apigenin treatment. Both Scramble and sgTAZ cells were treated with apigenin (0-64 μM) for 48 h, and cell viability was measured via the SRB assay. e Schematic diagram of the mechanism of apigenin-mediated antitumor activity by inhibiting YAP/TAZ activity in TNBC cells. Apigenin inhibited YAP/TAZ activity and decreased the expression of downstream genes by targeting YAP/TAZ-TEAD protein-protein interaction manufacturer's instructions. We performed real-time polymerase chain reaction (PCR) analysis using the Power SYBR Green PCR Master Mix Power (Roche, Mannheim, Germany) on a Bio-Rad CFX96 detection system. The mRNA levels of target genes were standardized to the level of β-Actin. Primer sequences (in the 5′-3′direction) are as follows: CTGF-F: GCAGAG CCGCCTGTGCATGG; CTGF-R: GGTATGTCTTCA TGCTGG; CYR61-F: CACACCAAGGGGCTGGAATG; CYR61-R: CCCGTTTTGGTAGATTCTGG; β-Actin-F: GGTGAAGGTCGGAGTCAACGG; β-Actin-R: GAGG TCAATGAAGGGGTCATTG.

Tumorigenic evaluation assays
MDA-MB-231 cells were pretreated with DMSO or 20 µM apigenin for 48 h. Living cells were quantified by using trypan blue exclusion assay. Cells were suspended in PBS containing 50% Matrigel (BD#354230). A series dilution of apigenin-treated and DMSO-treated cells (10 6 , 10 5 , 10 4 ) was injected into the right flank of female BALB/ c nude mice (5-6-weeks-old; Charles River, Beijing, China). Tumor formation efficiency and tumor sizes were recorded once a week. Tumor volumes were calculated according to the formula of Length × Width 2 /2. The care and use of animals were approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Guangzhou, China).

Statistical analysis of data
GraphPad Prism 5 was used for statistical analyses. Student's t-test was used for comparison between groups. Data are expressed as mean ± SD. Statistical significance was set at P < 0.05.