BAI1 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer via Modulating p73 Transactivation

Background: Brain-specic angiogenesis inhibitor 1 (BAI1) which belongs to putative G-protein-coupled receptors (GPCRs), has been found down-expressed in various cancers and involved in cancer pathogenesis. However, the role and underlying mechanisms of BAI1 in triple negative breast cancer (TNBC) are still unclear. Methods: The expression levels of BAI1 in TNBC samples and cell lines were examined by immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting (WB). The functional effects of BAI1 on biological behaviors of TNBC cells were detected using plasmid and siRNA for BAI1 overexpression and knockdown, and the underlying mechanisms were investigated by Immunoprecipitation (IP), immunouorescence (IF) and luciferase reporter assay. Results: BAI1 was downregulated in TNBC tissues and was signicantly associated with poor disease-free survival. Functional experiments indicated that BAI1 inhibited cell proliferation and induced cell apoptosis and cell cycle arrest. Additionally, BAI1 overexpressed cells were more sensitive to cisplatin. Mechanistically, BAI1 interacted with MDM2, thereby enhanced p73 transcriptional activity, then promoted p21and BAX mRNA and protein expression. Overexpression of p73 abolished the BAI1 knockdown induced cell proliferation and the G2 phase cell population of TNBC, the sensitivity to cisplatin also rescued by overregulating p73 in BAI1 knockdown TNBC cells. Conclusions: Our results indicate that BAI1 is a promising prognostic factor in TNBC, and the expression of BAI1 inhibits cell proliferation and induces cell apoptosis and cell cycle arrest; Meanwhile, BAI1 increases the sensitivity of TNBC to cisplatin. For the underlying mechanism, BAI1 specically binds to MDM2, and exerts its anti-tumor function by affecting the transcriptional activity of p73 protein, then inhibits the malignant progression of TNBC. The BAI1/MDM2/p73 axis may represent a potential target in the future research for TNBC. cells by inhibiting cell cycle progression and inducing cell apoptosis. Moreover, increased TNBC cell sensitivity to cisplatin. Furthermore, interacts with then increases p73 transcriptional activity. further elucidate the interaction of BAI1 and MDM2 and test the combination of BAI1 inhibition and cisplatin to achieve an optimal effect in TNBC. Overall, our data suggested that BAI1 functioned as an anti-tumorigenesis protein in TNBC by interacting with MDM2 and increasing p73 transcriptional activity. Thus, BAI1 may represent a potential therapeutic target for the clinical intervention of the


Introduction
Breast cancer is the most commonly cancer and the leading cause of cancer death among females [1] . Triplenegative breast cancer (TNBC) is a breast cancer subtype that lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression, and accounts for 15-20% of all breast cancers [2] . Compared to other subtypes, TNBC patients usually have poorer survival due to lack of effective targeted therapeutics. Thus, further exploring the potential molecular mechanisms of TNBC is critical to improving clinical outcomes. BAI1, an orphan G protein-coupled receptor-type seven transmembrane protein, was initially identi ed in a screen for genes regulated by the tumor suppressor p53 [3] . Structurally, The C-terminus of BAI1 contains a QTEV motif that mediates binding to PDZ domain-containing proteins which associates with a number of scaffolding proteins involved in intracellular signaling [4] . The N-terminal of BAI1 can be cleaved at the GPS site and releases soluble 40 and 120 kDa fragments known as vasculostatins, which suppress angiogenesis in vitro and inhibit tumor growth in vivo [5,6] . Reduced BAI1 expression has been observed in several tumor types, including glioblastoma, Pulmonary adenocarcinomas, gastric and colorectal cancer [7][8][9][10] . Previous study showed that signi cant downregulation of BAI1 in primary breast cancers and was associated with unfavorable patient outcomes [11] . Currently, an emerging and promising therapeutic modality called oncolytic viral (OV) therapy has been introduced in several malignant tumors [12,13] . A recent study showed that delivering Vstat120 (extracellular fragment of BAI1) to highly malignant breast cancer cells using oncolytic virus (34.5ENVE) transport system has showed promising results to selectively kill these tumor cells which may help provide promising future prospect for use of BAI1 as human diagnostic and therapeutic tool [11] . Given the functional importance of BAI1 in tumorigenesis and malignance, and its clinical translational value, understanding the underlying mechanism of BAI1 regulation in TNBC should facilitate the development of new therapeutic agents for these patients.
The well-de ned p53 tumor suppressor protein, which plays an important role in preventing tumor development, has been found occurs somatic mutation in approximately 60% of TNBCs [14] . Whereas p73, a member of the p53 family, shares remarkable homology in DNA sequence and protein structure with p53, and transactivates most p53 transcriptional target genes by recruiting to p53-response elements within gene promoters, like p21, Bax, thereby induces cell cycle arrest and apoptosis in tumor cells [15][16][17] . Unlike p53, which is widely mutated in human cancers, mutations of p73 is rare in human cancers [18][19][20] . Furthermore, p73 forms an interaction with mouse double minute 2 (MDM2), an E3 ubiquitin ligase, which binds to the p53 N-terminal transactivation domain [21] , inhibiting its transcriptional activity [22] and triggering its degradation via ubiquitinproteasome pathways [23,24] . However, the interaction between MDM2 and p73 negatively regulates the transcriptional activity of p73 but slightly impacts its degradation which distinct from the mechanism used for p53 regulation [25,26] . Previous studies found that MDM2 deletion increased levels of p73, then induced apoptosis and G2 cell-cycle arrest, which compensates for the loss of p53 in p53-de cient cancers [27] . Therefore, p73 provides a legitimate and attractive target in cells lacking functional p53 and provides a promising strategy for cancer therapy.
Previous studies indicated that BAI1 prevents PSD95 and p53 polyubiquitination and degradation through an interaction with MDM2 which regulates synaptic plasticity and suppress medulloblastoma formation respectively [28,29] . Meanwhile, a great number of studies showed that MDM2 interacts with p73 and inhibits its transcriptional activity in p53 mutated tumors. Whether BAI1 excises its anti-tumor function by binding to MDM2 and thereby enhances the transcriptional activity of p73 in TNBC cells still needs study. Meanwhile, few studies focus on the chemosensitivity in uence of BAI1 in malignant cancers. Therefore, we examined the potential role of BAI1 in chemosensitivity of TNBC and evaluated whether the anti-tumor activity of BAI1 is modulated through interaction with the MDM2/p73 pathway.
In the present study, we investigated BAI1 expression and correlation with clinicopathologic features and prognosis in TNBC patients. Then exploited BAI1 function and its interaction with p73-MDM2 pathway.

Patients and samples
This study was approved by the Institutional Review Board of the Fudan University Shanghai Cancer Center, and informed consent was obtained from all patients. The expression of BAI1 in TNBC patients was detected using immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting (WB). A tissue microarray (TMA) with 120 para n-embedded TNBC samples were performed to identify the expression levels of BAI1 in TNBC patients. qRT-PCR was performed to examine BAI1 mRNA expression in 159 breast cancer with different subtypes, 63 paired TNBC tumor samples and adjacent normal tissues (ANTs). Western blotting was performed to examine BAI1 protein expression in eight paired TNBC tumor samples and ANTs.

Analysis of Publicly Available Datasets
To analyze mRNA expression of BAI1 in whole breast cancer samples and different subtypes, and the effect of BAI1 expression on the prognosis of patients with breast cancer, the Gene Expression Pro ling Interactive Analysis (GEPIA) online tool (http://gepia.cancer-pku.cn) were used to analyze mRNA expression of BAI1 and prognostic value in BC samples from TCGA provisional dataset. were maintained according to standard American Type Culture Collection (ATCC) protocols. All cell lines were cultured in high-glucose DMEM medium containing 10% FBS and 1% penicillin/streptomycin in a 37°C humidi ed incubator with 5% CO2.
Cisplatin (purchased from MCE) was suspended in dimethyl sulfoxide (DMSO). Cells were seeded in 96-well plates with 100 μl of medium at a concentration of 4000 cells/well. DMSO was used as the negative control. The cells were then cultured for 48h and then tested using a CCK-8 kit to measure the half maximal inhibitory concentration (IC50).

Immunohistochemistry (IHC)
Slides were dewaxed at 60 °C for 2 h followed by three washes with xylene and rehydrated with different concentration gradients of ethanol, then washed with distilled water for 10 min. For antigen retrieval, slides were put into the ethylene diamine tetraacetic acid buffer (EDTA, pH 6.0) for 4 min when the buffer was heated to 100 °C. Then inhibited the endogenous peroxidase activity by 3% hydrogen peroxide for 30 min. After that, the slides were incubated with anti-BAI1 antibody (1:100 dilution; Abcam, Shanghai, China) overnight at 4 °C.
The slices were incubated with a biotin-labeled secondary antibody for 30 min, then followed by 3, 3′diaminobenzidine (DAB; Zhongshan biotech) substrate.
RNA extraction and quantitative real-time PCR Cells were collected, and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription (RT) was conducted using PrimeScript RT Master Mix (Takara, Dalian, China) to synthesize cDNA. qRT-PCR was performed with SYBR Green PCR Master Mix (Takara, Dalian, China) on an ABI 7900HT (PE Applied Biosystems) qPCR machine. For relative quanti cation, target gene mRNA expression was normalized to GAPDH expression. The primers used in the qRT-PCR assays are listed in (Additional le 1: Table S2).

Western blotting
Cells were collected and lysed with RIPA buffer (Thermo Scienti c) supplement with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates was centrifugated at 12,000 g for 15 min at 4°C , the protein supernatants were collected and quanti ed using a bicinchoninic acid assay (Thermo Scienti c) according to the manufacturers' instruction, then proteins were subjected to SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, USA). after incubated with blocking buffer (5% non-fat dry milk in TBST) for 1 h, PVDF membranes were cultured in indicated primary antibodies overnight at 4℃. After three washes with TBST and incubated with HRP-conjugated secondary antibodies for 1 h, antibody detection was conducted using an enhanced chemiluminescent substrate kit (Yeasen).
RNA interference, plasmid transfection, and lentivirus transduction BAI1 siRNA sequences and negative controls were designed by GenePharma (Shanghai, China) and used to transfect cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The target sequences of BAI1 siRNAs are listed in (Additional le 1: Table S3). For overexpression, full-length BAI1 and p73 were identi ed and cloned to generate Flag-BAI1 and His-p73 constructs. To construct stable BAI1 overexpression cells, lentivirus was constructed and used to infect MDA-MB-231 and MDAMB-468 cells, which were then selected with puromycin.
Cell viability and colony-formation assay Cells were seeded in 96-well plates and cell viability was assessed using a Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. For colony-formation assays, 1000 viable cells were seeded in 6-well plates and cultured for 2 weeks. Colonies were xed with ethanol and stained with 1% crystal violet before being counted.

Flow cytometry analysis
Cell cycle and cell apoptosis analyses were performed via ow cytometry on a FACS can instrument (Beckman Coulter, Brea, CA, USA). For the cell cycle analysis, cells were harvested, and xed with 70% ethanol at 4 °C overnight, and stained with propidium iodide (50 μg/ml), containing 100 μg/ml RNase A for 15 min. For apoptosis analysis, cells were harvested, washed with PBS, and incubated with Annexin V-PE and 7-AAD (BD Biosciences, San Diego, CA, USA).

Nuclear cytoplasmic fractionation
The extraction and isolation of nuclear and cytoplasmic protein were performed by using the Nuclear

Results
BAI1 suppresses breast cancer progression and correlates with better prognosis BAI1 mRNA expression in TNBC samples was examined by speci c database and qRT-PCR, and the protein expression of BAI1 in TNBC samples was examined by IHC, and western blot. BAI1 mRNA was downregulated in BC samples compared with that in normal tissues (Fig.1a) and was signi cantly downregulated in TNBC samples compared with that in luminal subtype BC (Fig.1b) in TCGA breast cancer dataset. Importantly, BAI1 low expression was signi cantly correlated with poor OS in TCGA database (P= 0.025, Fig.1c). similarly, BAI1 mRNA was signi cantly downregulated in TNBC samples compared with other subtypes (P< 0.001, Fig1d) and that in the paired ANTs (P< 0.001, Fig.1e) in our cohort. Furthermore, we found that BAI1 protein expression was also downregulated in eight TNBC tumor samples compared with that in the paired ANTs (Fig.1f). The IHC results of 120 TNBC samples showed that BAI1 downexpression was associated with higher tumor burden, higher Ki67 index and increased risk of local recurrence (Additional le 2: Table 1). Kaplan-Meier survival analysis showed that BAI1 low staining were correlated with worse DFS and OS in TNBC (p = 0.008, p = 0.037,  Table 2). Taken together, downregulated of BAI1 is related to poor prognosis in TNBC patients. Representative BAI1 IHC stanning were showed in Fig. 1i.

BAI1 inhibits TNBC cell proliferation and induces cell apoptosis
To evaluate the potential effect of BAI1 on the biological behaviors of TNBC cells, basal BAI1 mRNA and protein expression were evaluated in six TNBC cell lines (Fig. 2 a, b). BAI1 expression was relatively low in MDA-MB-231 and MDA-MB468 cells which were selected for overexpression, while Hs-578t and BT549 cells with relatively high basal BAI1 expression were selected for knockdown. qRT-PCR and western blotting analyses were used to con rm the e ciencies of overexpression and knockdown (Fig. 2 (Fig. 3a,  b). Next, we examined the effect of BAI1 on cell-cycle progression by ow cytometry. Overexpression of BAI1 increased the S-phase cell distribution and decreased the cell distribution at G2-phase, whereas BAI1 knockdown decreased the S-phase cell population and increased the G2-phase cell population, indicating that BAI1 promotes the S/G2 cell arrest (Fig. 3c, d). Taken together, these results suggest that BAI1 plays an important anti-tumorigenic role in suppressing TNBC cell growth.

BAI1 overexpression results in enhanced tumor sensitivity to cisplatin
The expression of BAI1 is signi cantly associated with the cell cycle and cell death progression. BAI1 overexpression MDA-MB-231 and MDA-MB-468 cells were signi cantly more sensitive to cisplatin than the negative control cells (Fig. 4a). Consistently, BAI1 knockdown Hs578T and BT549 cells were more resistant to cisplatin than the vector control cells (Fig. 4a). Both colony formation (Fig. 4b, c) and CCK8 (Fig. 4d) assays indicated that BAI1 overexpression promoted the e cacy of cisplatin, while BAI1 knockdown decreased the e cacy of cisplatin in TNBC cells. These data suggest that BAI1 plays an important role in cisplatin sensitivity of TNBC.

BAI1 interacts with MDM2 and regulates the transcriptional activity of p73
We further examined whether MDM2/p73 axis plays a role in the molecular regulation by which BAI1 alters cell cycle and cell death progression. First, Western blotting revealed that key mediators of the cell cycle and apoptosis, namely Bax and p21 were positively regulated, while Cyclin A2, Cyclin B1, Bcl2 were negatively regulated by BAI1 (Fig. 5a). However, the mRNA and protein expression of p73 did not signi cantly affected by BAI1 in TNBC cells (Fig. 5b, c), Then we validated the interaction of BAI1 and MDM2 in TNBC cells. IP analysis with an anti-Flag antibody showed that exogenously expressed Flag-BAI1 speci cally interacted with endogenous MDM2 (Fig. 5d). Consistently, immuno uorescence (IF) staining experiments showed that BAI1 reduces the co-localization of MDM2 and p73 (Fig. 5e). Besides, cytoplasmic and nuclear fractions analysis showed that BAI1 expression increased the expression of p73 and reduced the content of MDM2 in the nucleus (Fig. 5f). These data con rmed that BAI1 interacts with MDM2 in TNBC and affect the interaction between MDM2 and p73. Luciferase assay was performed to evaluate to further investigate whether BAI1 regulates the transcriptional activity of p73 protein through interact with MDM2. The results showed that BAI1 overexpressing in MDA-MB-231 cells signi cantly induced the occupancy of p73 on the promoters of p21 and BAX (Fig. 5g). Furthermore, qRT-PCR was used to validate mRNA expression of the p73 target genes p21, Bax were upregulated or downregulated by overexpression or knockdown of BAI1 without affecting the expression of p73 in TNBC cells (Fig. 5h). Collectively, we demonstrate that BAI1 could interact with MDM2 and increases p73 transcriptional activity in TNBC.

BAI1 inhibits TNBC tumor proliferation in a MDM2/p73-mediated manner
To further elucidate whether BAI1 regulated TNBC cell-cycle and apoptosis progression and sensitivity to cisplatin by increasing p73 transcriptional activity, we transfected His-p73 plasmids into Hs578T and BT549 cells with BAI1 downexpression. We found that overexpression of p73 signi cantly abolished the positive effects of BAI1 knockdown on cell proliferation (Fig. 6 a-c). Apoptosis assay indicated that p73 overexpression reversed the inhibitory effects of BAI1 knockdown on cell apoptosis (Fig. 6d,e). Cell cycle analysis indicated that overexpression of p73 signi cantly abolished the negative effects of BAI1 downexpression on cell-cycle arrest (Fig. 6f,g). The resistance to cisplatin induced by BAI1 knockdowning in Hs578T and BT549 cells was also signi cantly abolished by p73 overexpression (Fig. 6h). Furthermore, the increased expression of Cyclin A2, Cyclin B1, Bcl2, and decreased expression of Bax, p21 induced by BAI1 downexpression was partly blocked by incubation with p73 overexpressing plasmids (Fig. 6i). These results provide evidence that the antitumorigenesis role of BAI1 is mediated through the regulation of p73 transcriptional activity via interaction with MDM2.

Discussion
In this study, we found that BAI1 expression was downregulated in TNBC, which correlated with poor DFS. Furthermore, we demonstrated that BAI1 induced cell apoptosis and suppressed tumor growth by increasing p73 transcriptional activity via interaction with MDM2 (Fig. 6j). In addition, BAI1 overexpressing increased the sensitivity to cisplatin in TNBC cells. Notably, there is an established body of research in several types of cancer showing that BAI1 is involved in tumorigenesis and patient prognosis. Reduced BAI1 expression has been observed in several tumor types, including glioblastoma, Pulmonary adenocarcinomas, bladder transitional cell carcinoma, gastric and colorectal cancer [7][8][9][10][30][31][32] . Immunohistochemistry staining indicated that lower levels of BAI1 contributed to poor prognosis of lung cancer patients [33] . Strikingly, previous study showed that signi cant downregulation of BAI1 in primary breast cancers and was associated with unfavorable patient outcomes [11] . Accordingly, our data suggested that BAI1 downexpression is associated with worse patient clinical outcomes, which is consistent with the previous study.
The investigation of the biological function of BAI1 in TNBC suggested that BAI1 suppressed TNBC cells proliferation. Meanwhile, cell apoptosis and cell cycle analysis also indicated that BAI1 could induce cell apoptosis and S/G2 cell cycle arrest. Ectopic BAI1 expression decreased Cyclin A2, Cyclin B1, and Bcl2 expression, but increased P21 and Bax expression. A recent research suggestted that BAI1 involved into the antitumorigenic effect of EPZ-6438 (Tazemetostat, an enhancer of zeste homolog 2 inhibitor), blocked medulloblastoma cell growth in vitro and in vivo, and prolonged survival in orthotopic xenograft models [34] .
Moreover, BAI1 has also been found play an important role in the antitumorigenic action of HspB2 (Heat shock protein B2), which effectively inhibited pancreatic cancer cell proliferation [35] . Lei Liu et al found that overexpressed of BAI1 dramatically inhibited lung cancer cell proliferation, migration, invasion, colony formation, and in vivo metastasis by inducing metabolic reprograming [33] . According to our data, which suggested that BAI1 acts as a tumor suppressor and involves in tumor growth of TNBC.
BAI1 was found involved into the inhibition effect of decylubiquinone on breast cancer growth and metastasis [36] . Increasing evidence showed that p73 is an important determinant of chemosensitivity in TNBC via activated cell death signaling pathways [37,38] , and a large variety of chemotherapeutic agents, such as camptothecin, etoposide, and cisplatin, can upregulate p73 expression [39] . Cisplatin is a chemotherapy agent that has been widely used in clinical trials targeting breast cancers. Here, we found that BAI1 expression is related to sensitivity to cisplatin of TNBC. Our ndings showed that BAI1 overexpression increased the sensitivity of MDA-MB-231 and MDA-MB-468 cells to cisplatin, while BAI1 knockdown in Hs578T and BT549 cells decreased the sensitivity to cisplatin. Taken together, these data suggest that BAI1 expression may be used to stratify TNBC patients for cisplatin therapy to enable more effective tailoring of chemotherapy.
Further investigations about the molecular mechanisms of BAI1 in malignant tumors con rmed the interaction between BAI1 and MDM2 [29] . Previous studies found that MDM2 deletion increased levels of p73 and induced apoptosis and G2 cell-cycle arrest, which compensates for loss of p53 in p53-de cient cancers [25,26,40,41] .
Reports have indicated that p73 plays a key role in cancer growth and progression via MDM2 regulation in p53 mutated breast cancers [27,42,43] . We observed that BAI1 had no signi cant in uence on the mRNA and protein expression of p73. The IP experiment con rmed the interaction between BAI1 and MDM2, and the IF analysis validated the decreasing interaction of p73 and MDM2 when overexpressing BAI1. As shown in our results, overexpressing of BAI1 could lead to increased expression of p73 target genes related to cell cycle progression and cell death signaling pathway. The luciferase assay con rmed that BAI1 overexpressing signi cantly induces the occupancy of p73 on the promoters of p21 and BAX, which supports our speculation that BAI1 can positively regulate p73 transcriptional activity by interacting with MDM2. To determine whether MDM2/p73 axis is a downstream target involved in BAI1-reduced TNBC growth and apoptosis suppression, we knocked in p73 in BAI1 downexpressing cells. The BAI1 downexpression-mediated induction of cell growth and reduction of apoptosis was dramatically inhibited when p73 was knocked in. These data suggest that p73 is a crucial downstream target of BAI1 that mediates BAI1-reduced cell growth and BAI1-induced cell apoptosis in TNBC cells.
In summary, our study indicated that BAI1 was downregulated in TNBC tissues and BAI1 downexpression was associated with high risk of local recurrence and poorer clinical outcomes in TNBC patients. BAI1 suppressed TNBC cells growth by inhibiting cell cycle progression and inducing cell apoptosis. Moreover, BAI1 overexpressing increased TNBC cell sensitivity to cisplatin. Furthermore, BAI1 interacts with MDM2, then increases p73 transcriptional activity. It is important to further elucidate the interaction of BAI1 and MDM2 and test the combination of BAI1 inhibition and cisplatin to achieve an optimal effect in TNBC. Overall, our data