Formononetin enhances the chemosensitivity of triple negative breast cancer via BTB domain and CNC homolog 1-mediated mitophagy pathways

This study aimed to investigate the effects of formon-onetin on triple negative breast cancer (TNBC). Clinical samples were collected from patients with TNBC. Overall survival rates were evaluated using the Kaplan-Meier method. Gene expression was determined using immunohistochemistry, immunofluorescence and western blot. Cellular functions were determined using CCK-8, colony formation and propidium iodide (PI) staining. Xenograft assay was performed to further verify the effects of formononetin (FM) on TNBC. We found that FM combined therapy suppressed the metastasis of TNBC and increased the overall survival rates of TNBC patients. Moreover, FM suppressed the proliferation and induced mitochondrial damage and apoptosis of TNBC cells. FM increased the expression of the BTB domain and CNC homolog 1 (BACH1) in TNBC tissues as well as cells. However, BACH1 knockdown antagonized the effects of FM and promoted the survival of TNBC cells. FM suppressed the tumor growth of TNBC. Taken together, FM suppressed the aggressiveness of TNBC via BACH1/p53 signaling. Therefore, FM may be an alternative strategy for TNBC.


INTRODUCTION
Breast cancer is the most common tumor among women worldwide (Braden et al., 2014;Burstein et al., 2019). Recent decades witness an increase in the incidence and mortality of breast cancer in China (Ding et al., 2020;Fan et al., 2014). Triple negative breast cancer (TNBC) is characterized by a high proliferative index, histological grade, and metastatic states (Garrido-Castro et al., 2019). Although great advances have been made in chemotherapy, radiotherapy, and surgery for TNBC (Bianchini et al., 2016). However, the overall survival rates of TNBC are still unsatisfactory (Newman and Kaljee 2017). The high recurrence and metastatic properties of TNBC neutralize the clinical outcomes . Therefore, a new strategy for TNBC is urgently needed.
Traditional Chinese medicine (TCM), with high efficiency and few side effects, is widely applied in the treatment of breast cancer (Chan et al., 2021;Yang et al., 2021). Formononetin (FM), extracted from astragalus membranaceus and spatholobus suberectus and with anti-inflammatory and anti-carcinogenic properties, is used as adjuvant therapy for breast cancer Xin et al., 2019;Yu et al., 2020). Previous studies reveal that formononetin exerts its anti-cancer function via modulating several signaling. For instance, formononetin suppresses the chemoresistance of TNBC via inactivating autophagy . Formononetin induces prostate cancer cell mitochondrial and apoptosis via regulating IGF-1/IGF-1R pathways (Huang et al., 2013). Additionally, formononetin inhibits the immune suppressiveness of cervical cancer through inactivating MYC/STAT3/ PD-L1 signaling (Wang et al., 2022). This study explored the effects of NP (vinorelbine and cisplatin) combined with formononetin on TNBC.

Patients
Clinical samples were collected from patients with refractory TNBC undergoing chemotherapy (vinorelbine and cisplatin, NP) with or without FM. at People′s Hospital of Dongtai City from April 1, 2019 to March 31, 2021. The samples were immediately stored in liquid nitrogen at -80°C. This study was approved by the Ethical Committee of People′s Hospital of Dongtai City. All patients signed confirmed consent. The inclusion criteria are: (1) the patients diagnosed with mTNBC; (2) women under the age of 70; (3) no serious complications occurred after the operation; (4) general condition score: ECoG 0-2; (5) patients in the combined group insisted on taking formononetin for at least 8 months. The exclusion criteria are: the mTNBC patients with an estimated ≤3 months of survival time; (2) patients accompanied by severe impairment or insufficiency of heart, liver and kidney functions; (3) patients with poor compliance and unable to adhere to treatment.

MDA and SOD determination
The release of MDA and SOD was determined using specific commercial kits (Beyotime, Shanghai).

CCK-8 assay
After 48-hour transfection, cells were collected. Then cells were plated into 24-well plates and cultured for 0, 12, 24, and 48 h. After being supplemented with CCK-8 regents and cultured for another 2 h, cells were detected by a microplate reader at the wavelength of 450 nm.

Colony formation assay
After transfection, cells were plated into a 24-well plate. After 2 weeks of culture, cells were fixed and stained with 0.1% crystal violet. Subsequently, the colonies were visualized using a microscope.

Propidium iodide (PI) staining
After transfection, cells were plated into a 24-well plate. Then cells were treated with PI solution (2 µg/ mL). Finally, PI positive cells were captured by a fluorescence microscope (Leica, Germany).

Xenograft assay.
18 BALB/c nude mice (6-8 weeks, 18-22 g) were purchased from the Animal Center of Nanjing Medical University. Mice were randomly divided into three groups: control group, NP+FM group, and NP+FM+CDDO-ME (CDDO-ME) group. Each mouse was inoculated subcutaneously with 3×10 4 cells. The tumor was measured every three days. Tumor size was calculated as followed: V=lw 2 /2. At 21 days, mice were euthanized, and tumor were collected. This study was authorized by the Animal Care Broad of People's Hospital of Dongtai City.

Statistical analysis
All data were analyzed using SPSS 20.0. The difference was analyzed using the Student t-test and ANOVA assay. The survival rates of patients were analyzed using Kaplan Meier and log-rank test. P<0.05 was deemed as a significant difference.

The characteristics of TNBC patients
As shown in Table 1, the participants were all female. 68% of the TNBC patients had increased LDH levels. 45% showed visceral metastasis and 33% with recurrence. Additionally, combined therapy significantly improved the overall survival rate of TNBC patients (Fig. 1).

FM promotes oxidative stress and mitochondrial damage in TNBC
Previous studies reveal that FM suppresses the progression of multiple myeloma via inducing oxidative stress. Then we determined the release of oxidative stress in TNBC. FM enhanced the effects of NP on the release of MDA and GSH (Figs 2A and B). FM enhanced mitochondrial aggregation induced by NP (Fig.   2C). Moreover, FM+NP markedly increased cyto C protein expression compared with the NP group (Fig. 2D).

FM suppresses the aggressiveness of TNBC cells.
To further verify the effects of FM on TNBC, we determined the MCF7 cellular functions. Compared to the control group, FM treatment significantly suppressed the cell viability of MCF7 cells (Fig. 3A). This was consistent with the results from the colony formation assay. FM markedly inhibited the proliferation of MCF7 cells (Fig. 3B). Additionally, FM significantly enhanced the apoptosis of MCF7 cells (Fig. 3C). FM remarkably increased the protein expression of BAX and Caspase3 and decreased Bcl2 (Fig. 3D).

FM increases BACH1 expression
BACH1 is evidenced to play a vital role in mitochondrial function. We then determined the potentials of BACH1 in TNBC. The online database showed that BACH1 expression was decreased in invasive breast cancer tumors (Fig. 4A). To further verify this, we determined BACH1 expression in TNBC patients. As shown in Fig. 4B, BACH1 expression in patients administrated with NP+FM. Moreover, the protein expression of BACH1 was markedly increased in cells treated with NP+FM (Figs. 4C and D).

BACH1 transmits ROS signaling to mitochondria
BACH1 suppresses the aggressiveness of cancer cells via increasing the release of mitochondrial ROS (Hao et al., 2021). We, therefore investigated the potentials of BACH1 mitochondrial ROS. Figure 5A showed the transcription efficiency of sh-BACH1. BACH1 knockdown antagonized the effects of FM and increased the cell ability of MCF7 cells (Fig. 5B). Moreover, BACH1 knockdown suppressed the protein expression of cyto-C, and cleaved caspase3 and -9 (Fig. 5C). Additionally, BACH1 knockdown suppressed mitochondrial aggrega-      (Fig. 5D). The apoptosis rates were significantly decreased in the sh-BACH1 group (Fig. 5E). To further confirm the roles of BACH1 in mitochondrial ROS. Cells were exposed to NAC (an ROS inhibitor). As shown in Fig. 5F, NAC antagonized the effects of FM and decreased the protein expression of BACH1.

FM regulates mitophagy via inducing BACH1-mediated activation of p53
We further investigated the potential underlying mechanisms. The online database STING predicted the po-tential genes interacting with BACH1 (Fig. 6A). Then we found that BACH1 may regulate the expression of p53. To further verify this, cells were treated with FM and/ or sh-p53. As shown in Figs 6B and C, FM promoted p53 translocation from cytoplasm to mitochondria. p53 deficiency suppressed protein expression of cyto C and Caspase9 mitochondrial aggregation (Fig. 6D) as well as mitochondrial aggregation (Fig. 6E). Moreover, p53 knockdown decreased the protein expression of PINK1, PARK2 (Fig. 6F). p53 knockdown promoted the cell  viability and inhibited the apoptosis of MCF7 cells (Figs 6G and H).

FM suppresses the tumor growth of TNBC via regulating BACH1
To further verify the effects of FM on TNBC, in vivo assay was performed. As shown in Fig. 7A-C, FM suppressed the tumor size, volume and weight, which was abated by BACH1 deficiency.

DISCUSSION
In this study, FM suppressed improved the clinical outcome of TNBC patients. FM induced oxidative stress and apoptosis of MCF7 cells suppressed tumor growth. Additionally, FM suppressed mitophagy via inactivating BACH1/p53 signaling pathways.
Formononetin (FM) possesses anti-tumor properties in various cancers (Tay et al., 2019). For instance, FM suppressed the proliferation and metastasis of ovarian cancer cells (Zhang et al., 2018). FM suppresses the growth and migration of gastric cancer . In breast cancer, FM induced breast cancer cell cycle rest (Chen et al., 2011). FM inhibits Taxol-chemoresistance of breast cancer cells via suppressing autophagy signaling . However, seldom study focuses on the mitochondrial functions in breast cancer cells. In this study, FM transmitted ROS to mitochondria to release cyto C and induced the cascades of caspase-3 and caspase-9, the activation of which induced the apoptosis of MCF7 cells. These findings suggest that FM may exert its anti-tumor functions via impeding the mitochondrial function in breast cancer cells.
BACH1, a member of the cap'n'collar (CNC) b-Zip family, plays a key role in regulating oxidative stress (Wiel et al., 2019). BACH1 is a key regulator of mitochondrial metabolisms in cancer (Lignitto et al., 2019). For instance, BACH1 knockdown induces mitochondrial respiration and increases the chemosensitivity of papillary thyroid cancer cells to metformin (Yu et al., 2022). Moreover, BACH1 is overexpressed in TNBC patients and High levels of BACH1 predict poor overall survival and disease-free survival rates (Ou et al., 2019). Overexpressed BACH1 inhibits glycolysis as well as suppresses lactate catabolism in the tricarboxylic acid (TCA) cycle, and promotes breast cancer bone metastasis Padilla et al., 2022). These studies dictate that BACH1 may function as an oncogene in breast cancer. In this study, BACH1 was overexpressed in TNBC patients and MCF7 cells. Moreover, overexpressed BACH1 suppressed the release of oxidative stress and cancer cell apoptosis. Moreover, BACH1 alleviated FM-induced mitochondrial damage.
Mitochondrial metabolism plays a vital role in mitochondrial function, requiring intensive integration of mitochondrial morphology and dynamics (Chan, 2020). Mitophagy, erasing damaged mitochondria via autophagy, is a key process to maintaining mitochondrial quality (Srinivasan et al., 2017). However, dysfunction of mitophagy may induce the pathogenesis of cancer, such as hepatocellular carcinoma, colon as well as breast cancer (Chen et al., 2019;Deng et al., 2021;Yin et al., 2021). Therefore, to unraveling the underlying mechanisms may provide a novel target for cancer therapy. In this study, FM treatment induced the overexpression of p53. As a tumor suppressor, p53 suppresses tumorgenesis via inducing apoptosis, pyroptosis, ferroptosis as well as autophagy. In this study, FM stimulated p53/PINK1/PARK2 signaling pathways via inactivating BACH1, which promoted the release of cyto C and caspases as well as mitophagy-mediated mitochondrial dysfunction.
However, there are several limitations to this study. A large number of patients could make the results more convincing. Therefore, future studies will recruit more metastatic TNBC patients. Additionally, mitochondrial damage and mitophagy may induce other forms of death, such as ferroptosis and pyroptosis. Whether FM induced ferroptosis or pyroptosis. This needs further study.
In conclusion, FM improved the clinical outcomes of TNBC patients. Additionally, FM suppressed the aggressiveness of TNBC via regulating BACH1 signaling. Therefore, FM may be an alternative strategy for TNBC.