Naringenin Regulates FKBP4/NR3C1/TMEM173 Signaling Pathway in Autophagy and Proliferation of Breast Cancer and Tumor-Inltrating Dendritic Cell Maturation

TMEM173 is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits cGAS related signals that activate host innate immune responses. It has also been found to be involved in tumor immunity and tumorigenesis. Bc-GenExMiner, PROMO and STRING database were used for analyzing clinical features and interplays of FKBP4, TMEM173 and NR3C1. Transient transfection, western blotting, quantitative real-time PCR, luciferase reporter assay, immunouorescence and nuclear and cytoplasmic fractionation were used for regulation of FKBP4, TMEM173 and NR3C1. Both knockdown and overexpression of FKBP4, TMEM173 and NR3C1 were used to analyze effects on autophagy and proliferation of breast cancer (BC) cells. Flow cytometry analysis, cytokine analysis and exosome isolation and identication were utilized to test tumor-inltrating dendritic cell (TIDC) maturation. TMEM173 in basal-like subtype of BC patients, r= -0.25, p<0.0001, No=506. e Representative western blot analysis results of FKBP4, TMEM173 and endogenous control GAPDH. Western blot analysis showed a negatively correlated expression of FKBP4 and TMEM173 in BC and normal breast cells. f Representative western blot analysis results of FKBP4, TMEM173 and endogenous control GAPDH. Western blot analysis showed decreased FKBP4 expression and increased TMEM173 expression in T47D and BT549 cells treated by 100 nM naringenin for 24 h. g Representative western blot analysis results of FKBP4, TMEM173 and endogenous control GAPDH. Western blot analysis showed silencing FKBP4 resulted in upregulation of TMEM173 in T47D and BT549 cells. h Representative western blot analysis results of FKBP4-HA, TMEM173 and endogenous control GAPDH. Western blot analysis showed overexpressing FKBP4 resulted in downregulation of TMEM173 in T47D and BT549 cells. i RT-qPCR showed silencing FKBP4 resulted in upregulation of TMEM173 in T47D and BT549 cells (n=3 independent biological replicates). j RT-qPCR showed overexpressing FKBP4 resulted in downregulation of TMEM173 in T47D and BT549 cells (n=3 independent biological replicates). k Luciferase reporter assay showed silencing FKBP4 resulted in increased TMEM173 promoter activity in T47D and BT549 cells (n=3 independent biological replicates). l Luciferase reporter assay showed overexpressing FKBP4 resulted in decreased TMEM173 promoter activity in T47D and BT549 cells (n=3 independent biological replicates). *p<0.05, **p<0.01, ***p<0.001. analysis silencing NR3C1 in downregulation of TMEM173, overexpressing NR3C1 in upregulation of TMEM173 in T47D and BT549 cells. g, h RT-qPCR silencing NR3C1 resulted in downregulation of TMEM173, overexpressing NR3C1 resulted in upregulation of TMEM173 in T47D and BT549 cells (n=3 independent biological replicates). i, j Luciferase reporter assay showed silencing NR3C1 resulted in decreased TMEM173 promoter activity, overexpressing NR3C1 resulted in increased TMEM173 promoter activity in T47D and BT549 cells (n=3 independent biological replicates). k Representative western blot analysis results of NR3C1, FKBP4, TMEM173 and endogenous control GAPDH. Western blot analysis showed knockdown of NR3C1 by shRNA attenuated siFKBP4-upregulated TMEM173 expression in T47D and BT549 cells. l RT-qPCR showed knockdown of NR3C1 by shRNA attenuated siFKBP4-upregulated TMEM173 expression in T47D and BT549 cells (n=3 independent biological replicates). m Luciferase reporter assay showed knockdown of NR3C1 by shRNA attenuated siFKBP4-upregulated TMEM173 promoter activity in T47D and BT549 cells (n=3 independent biological replicates). *p<0.05, **p<0.01, axis is involved in naringenin-induced autophagy. a Representative blot analysis results of P62, Beclin1, FKBP4, LC3B and endogenous control GAPDH. Western blot analysis showed silencing FKBP4 resulted in downregulation of P62 and upregulation of Beclin1 and LC3B-II/LC3B-I in T47D and BT549 cells. b Representative western blot analysis results of P62, Beclin1, TMEM173-Flag, LC3B and endogenous control GAPDH. Western blot analysis showed overexpressing TMEM173 resulted in downregulation of P62 and upregulation of Beclin1 and LC3B-II/LC3B-I in T47D and BT549 cells. c Representative western blot analysis results of P62, Beclin1, FKBP4-HA, LC3B and endogenous control GAPDH. Western blot analysis showed overexpressing FKBP4 resulted in upregulation of P62 and downregulation of Beclin1 and LC3B-II/LC3B-I in T47D and BT549 cells. d Representative western blot analysis results of P62, Beclin1, TMEM173, LC3B and endogenous control GAPDH. Western blot analysis showed silencing TMEM173 resulted in upregulation of P62 and downregulation of Beclin1 and LC3B-II/LC3B-I in T47D and BT549 cells. e, f Representative western blot analysis results of P62, Beclin1, TMEM173, LC3B and endogenous control GAPDH. Western blot analysis showed knockdown of TMEM173 by shRNA attenuated naringenin-induced autophagy in T47D and BT549 cells treated by 100 nM naringenin for 24 h. g, h Measurement of autophagy ux in T47D and BT549 cells co ‐ f


Abstract
Background TMEM173 is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits cGAS related signals that activate host innate immune responses. It has also been found to be involved in tumor immunity and tumorigenesis.

Methods
Bc-GenExMiner, PROMO and STRING database were used for analyzing clinical features and interplays of FKBP4, TMEM173 and NR3C1. Transient transfection, western blotting, quantitative real-time PCR, luciferase reporter assay, immuno uorescence and nuclear and cytoplasmic fractionation were used for regulation of FKBP4, TMEM173 and NR3C1. Both knockdown and overexpression of FKBP4, TMEM173 and NR3C1 were used to analyze effects on autophagy and proliferation of breast cancer (BC) cells. Flow cytometry analysis, cytokine analysis and exosome isolation and identi cation were utilized to test tumorin ltrating dendritic cell (TIDC) maturation.

Results
In this study, we rstly identi ed that FKBP4/NR3C1 axis was a novel negative regulator of TMEM173 in BC cells. The effect of FKBP4 appeared to be at the transcriptional level of TMEM173 since it could suppress the promoter activity of TMEM173, thereby affecting TMEM173 at mRNA and protein levels. Bioinformatics and in vitro experiments further demonstrated that FKBP4 regulated TMEM173 via regulating nuclear translocation of NR3C1. We then reported that naringenin, a avonoid, could enhance autophagy and suppress proliferation of BC cells through the induction of TMEM173 in vitro and in vivo. Naringenin was also found to promote TIDC maturation through FKBP4/NR3C1/TMEM173 axis of both BC cells exosome and DC itself.

Conclusion
We demonstrated that naringenin could induce cell proliferation inhibition and cytoprotective autophagy of BC cells and enhance TIDC maturation, at least in part, though regulation of FKBP4/NR3C1/TMEM173 signaling pathway. Identi cation of FKBP4/NR3C1 axis as a novel TMEM173 regulator would provide insights for novel anti-tumor strategy against BC among tumor microenvironment.

Background
Breast cancer (BC) is a leading cause of cancer-related deaths in women aged 40 years and younger [1]. Early detection and comprehensive treatments, which consist of surgery, radiation, chemotherapy, endocrine therapy and targeted therapy, have dramatically improved the prognosis of BC patients. In recent years, immunotherapy in BC showed promising future. Cancer vaccines, bispeci c antibodies, and immune checkpoint inhibitors are veri ed to have potential applied value in BC immunotherapy [2]. For instance, adaptive immune checkpoint therapies by targeting cytotoxic T-lymphocyte antigen-4, programmed cell death-1 (PD-1) and ligand partner for PD-1 (PD-L1) for BC have been used in clinical trial [3,4]. Nevertheless, a portion of BC patients still cannot bene t from above-mentioned immunotherapy strategies [5]. Therefore, unravelling the potential molecular mechanisms of both innate and adaptive immune system in BC cells is essential to further understand and improve immune related anti-tumor effects.
Our previous team's study has discovered that HIV-2/SIV Vpx acts as a novel inhibitor of innate immune activation associated with TMEM173 signalosomes [17], we are wondering whether and how TMEM173 performs its anti-tumor effects by connection with novel molecular chaperones in BC.
As one of the most extensively studied proteins among the 18 identi ed human FK506-binding proteins (FKBPs), FK506-binding protein 4 (FKBP4), also known as FKBP52, has been reported to exhibit multiple functions which involve binding to different cellular receptors or targets in various kinds of cancers [18][19][20][21][22]. For example, FKBP4 has been demonstrated to interact with heat shock protein 90 (Hsp90) to affect steroid hormone receptor function in BC [23]. In terms of immune regulation, phytanoyl-CoA alphahydroxylase (PAHX) has been regarded as a speci c target of FKBP4 for studying the cellular signaling pathway in the presence of immunosuppressant drugs [24]. Our previous work found that FKBP4 interacted with non-coding RNAs and mRNAs during the occurrence and development of BC, thus playing a role in promoting cancer [22,25]. Nevertheless, current immunologic mechanism of FKBP4 is still in its infancy for BC, it is necessary to explore more new detailed contents of its regulation of innate and adaptive immunity functions during the occurrence and development of BC.
The NR3C1 (nuclear receptor subfamily 3, group C, member 1/glucocorticoid receptor) normally resides in the cell cytoplasm, the NR3C1 protein translocates to the nucleus when bound to glucocorticoids, thus involved in growth, reproduction, metabolism, immune and in ammatory reactions, as well as central nervous system and cardiovascular functions and tumor cellular proliferation and differentiation [26].
Researches on NR3C1 and BC have also been conducted in recent years, e.g., high levels of NR3C1 expression and high concentrations of cortisol have been shown to have an anti-proliferative effect in cancerous breast tissue [27]. Some studies have preliminarily found that NR3C1 is associated with FKBPs, but the speci c mechanisms remain unclear in BC [28].
Exosomes, also termed small extracellular vesicles, have a diameter ranging from 40 to 150 nm, which are secreted by different types of cells and contain various cargoes, including DNA, mRNA, noncoding RNA, proteins and so forth [29]. Currently, researches on the role of exosomes involved in cancer progression have grown exponentially, including immune regulation, which suggest that cancer cells could secrete large numbers of exosomes to regulate innate and adaptive immune cells among tumor microenvironment for immune escape [30]. For instance, a study by Möller et al. showed that highly metastatic murine BC cells derived exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity [31]. Although Demaria et al. identi ed exosome as a mechanism whereby DNA was transferred from irradiated BC cells to tumor-in ltrating dendritic cell (TIDC) [32], whether and how FKBP4 or TMEM173 participated in exosomes secreted by BC cells might regulate DC maturation and function remain to be explored.
In this study, we showed that naringenin, a avonoid shown anti-tumor effects in various carcinomas in other studies [33], promoted autophagy and suppressed proliferation via new-found

Gene silence
To validate hits from the genetic screens, breast cancer cells were transduced with pLKO.1 vectors, which in addition to the shRNA cassette carried a puromycin resistance cassette (pLKO.1-puro, Addgene plasmid #10878). The shRNAs against TMEM173 and NR3C1 were cloned into pLKO.1 vectors using the Age1 and EcoR1 restriction sites. The shRNA targeting sequences of TMEM173[8] and NR3C1 [34] were from published articles, lentiviral particles were produced as follows. In brief, HEK293T packaging cells were

Western blotting and co-IP
Knockdown e ciencies and biochemical responses were analyzed by western blotting. Cells were lysed in RIPA lysis buffer (EMD Millipore Corp.), supplemented with protease inhibitor and phosphatase inhibitor cocktail tablets (Roche). Separated proteins were transferred to nitrocellulose lter membranes and blocked in 5% milk in Tris-buffered saline, with 0.05% Tween-20. Immunodetection was done with various primary antibodies. Appropriate horseradish peroxidase-conjugated secondary antibodies were used and signals were visualized with enhanced chemiluminescence (Proteintech) by Tanon-5200 Chemiluminescent Imaging System (Tanon, China). Cells for co-IP were lysed in lysis buffer (50 mM Tris, pH 7.5, with 150 mM NaCl, 0.5% NP-40, and protease inhibitor and phosphatase inhibitor cocktail tablets (Roche) at 4°C for 30 min. After sonication and centrifugation, cell lysates were incubated with beads (Sigma) at 4°C overnight on a rotator. After six washes with wash buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween-20, 0.1 mM EDTA), 50 µL of elution buffer (100 mM glycine-HCl, pH 2.5) was added to resuspend the beads, and the eluted proteins were obtained by centrifugation, followed by SDS-PAGE and immunoblotting analysis.

Plasmids
TMEM173-Flag, VR1012-HA, the control plasmid Renilla luciferase vector and pGL3-Basic luciferase vector were kindly provided by Xiao-Fang Yu (Zhejiang University, China), FKBP4 (GenBank accession number 2288) and NR3C1 (GenBank accession number 2908) were cloned into the VR1012-HA vector using the SalI and BamHI sites. The TMEM173 luciferase reporter plasmid was constructed by Shanghai Generay Biotech Co., Ltd.

Luciferase reporter assay
Breast cancer cells were plated into 12-well dishes and transfected the following day. 1 µg of the reporter plasmid for TMEM173 promoter, 50 ng the Renilla luciferase control plasmid, and the indicated amounts of the expression plasmids were used per well. At 24 h posttransfection, luciferase activities were then measured by using a Dual-Luciferase reporter assay system (Promega) according to the manufacturer's instructions. Fire y luciferase activity was normalized to Renilla luciferase activity. Finally, the relative luciferase activities were expressed as fold changes over the empty-plasmid-transfected controls.

Immuno uorescence
T47D or BT549 cells grown on coverslips were xed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min in 0.1% Triton X-100 in PBS, and blocked using 5% BSA for 1 h. The cells were then incubated with primary antibodies at 4°C overnight. After a rinse with PBS, the cells were incubated with uorescent-conjugated secondary antibodies for 1 h at 37°C. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were captured using a ZEISS laser scanning confocal microscope (LSM710; Zeiss). ZEISS ZEN Microscope software was used for acquisition.

Nuclear and cytoplasmic fractionation
T47D or BT549 cells were transfected with the indicated siRNAs for 72 h, then cells were harvested, and the nuclear and cytoplasmic fractions were separated using Thermo Fisher Scienti c NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833) according to the manufacturer's protocol.

Transient transfection
Breast cancer cells cultured in 12-well tissue culture plates were transiently transfected with plasmids using Lipofectamine® 2000 Reagent (Invitrogen) or siRNAs using Lipofectamine® RNAiMAX Reagent (Invitrogen) as instructed by the manufacturer. The siRNA targeting FKBP4, NR3C1 and TMEM173, as well as negative control siRNA were purchased from RIBBIO (Guangzhou, China). 72 h later, the whole-cell extract was prepared for RT-qPCR or western blot analysis.

Cell proliferation assay
Cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8) (DOJINDO). All cells were seeded into 96-well plates at a density of 5000 cells/well in a 100 µl volume and incubated at 37°C under 5% CO 2 for 24, 48, 72 h, followed by the addition of 10 µl of CCK-8 solution. The absorbance in each well was measured after 1 h incubation using a microculture plate reader at a test wavelength of 450 nm. Three replicate wells were set up in each group, and three independent experiments were performed.

Colony formation assay
Five hundred cells per well of breast cancer were seeded in a 6-well plate for colony formation assay. Two weeks after, they were xed with 4% paraformaldehyde and stained with Crystal Violet. Colonies were quanti ed using ImageJ software.

Flow cytometry analysis
Single cell suspensions were surface-labeled using the mAbs mentioned above for 30 min at 4°C. After using Flow cytometry Staining buffer (MULTI SCIENCES, China), cells were analyzed with a BD FACSCalibur Flow Cytometer (Becton Dickinson). Cytometry data was analyzed using FlowJo software version 10.
Cytokine concentrations are expressed as pg/ml.

In vivo tumor xenograft assays
Six-week-old male BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co, Ltd (Beijing, P.R. China). Animal experimental procedures were approved by the Medical Ethics Committee of Shandong University (ECAESDUSM 2014056). The six-week-old male mice were randomized into different groups. T47D shControl and T47D shTMEM173 cells (5 × 10 6 cells/mice) were implanted subcutaneously into the ank of nude mice. After tumor formation, naringenin (50 mg/kg) was administered orally to its respective treatment groups of animals for 28 days. Tumor volume (mm 3 ) was measured every three days and calculated by the formula (length × width × width)/2. When the tumors had reached a volume of approximately 600 mm 3 , the mice were euthanized, and the tumors were excised and embedded in para n for IHC analysis.

Exosome isolation and identi cation
BC cells were cultured in exosome-depleted (160000 g, 16 h) complete medium, and the supernatant was collected after 72 h. After three successive centrifugations at 300 g (5 min), 1200 g (20 min) and 10000 g (30 min) at 4°C, exosomes were puri ed with Amicon Ultra-15 centrifugal lters with Millex-GP syringe membrane lters with a 0.22 µm pore size (Millipore Express) to eliminate debris and then centrifuged at 100000 g (70 min at 4°C). Vesicles resuspended were loaded onto the top of a step gradient comprising layers of 0.25, 0.5, 1.0, 1.25 and 2 M sucrose (S9378, Sigma). The gradients were centrifuged at 100000 g for 2.5 h using a Beckman SW41Ti rotor. Ten fractions of 1 ml were collected and separately washed with PBS at 100000 g for 2.5 h using a Beckman MLS50 rotor. The protein content of the exosomes was determined by using a BCA protein assay kit (Pierce, Pierce County, WA). The isolated exosomes were diluted in PBS and analyzed using a Nanoparticle Tracking Instrument (Particle Metrix, Germany) according to the manufacturer's protocol. The size and concentration of exosomes were analyzed using the Zetaview Analysis software.

Statistics
Two-tailed student's t-test was used in this study. Data shown was mean ± SD from at least three independent experiments. Statistical probability was expressed as *p < 0.05, **p < 0.01, ***p < 0.001.

Negative correlation of FKBP4 and TMEM173 in breast cancer
Firstly, we used bc-GenExMiner v4. 4[36], an online public tool focused on BC, to explore the clinicopathological characteristics of FKBP4 and TMEM173. Upregulated FKBP4 was found signi cantly related to luminal A, luminal B and basal-like subtype of BC patients than the normal group ( Figure 1A), and downregulated TMEM173 was signi cantly related to luminal A, luminal B and basal-like subtype patients than the normal group ( Figure 1B). Furthermore, we validated a signi cant negative association between FKBP4 and TMEM173 both in luminal A and basal-like subtype of BC patients ( Figure 1C, D), but not in luminal B and HER2-positive subtype of BC patients ( Figure S1). Then, the prognostic merits of FKBP4 and TMEM173 in luminal A and basal-like subtype of BC patients were further analyzed by using bc-GenExMiner v4.4, the Kaplan-Meier curve showed that increased levels of FKBP4 and decreased levels of TMEM173 were strongly correlated with worse overall survival both in luminal A and basal-like subtype of BC patients ( Figure S2).
We also tested ve BC cell lines (MCF7, T47D, BT549, BT474 and SKBR3) as well as normal breast cell line (MCF10A) for expression levels of FKBP4 and TMEM173. As shown in Figure 1E, expression of FKBP4 and TMEM173 were negatively correlated in BC cell lines, especially in T47D (representing luminal A subtype of BC) and BT549 (representing basal-like subtype of BC) cell lines. Since naringenin has been reported involved in immunity regulation [37], we wondered its effect of pharmacologically modulating TMEM173 expression. As shown in Figure 1F, naringenin signi cantly decreased FKBP4 expression and increased TMEM173 expression in T47D and BT549 cells.

FKBP4 negatively regulates TMEM173 at protein, mRNA and transcription level
To con rm the speci c interaction between FKBP4 and TMEM173 at the molecular level, we rstly used siRNA speci cally targeting FKBP4 in T47D and BT549 cells, which led to upregulation of TMEM173 at protein level ( Figure 1G). In addition, we transfected FKBP4-HA plasmid in T47D and BT549 cells, which resulted in downregulation of endogenous TMEM173 ( Figure 1H). These results clearly indicated that FKBP4 had a role in negatively regulating TMEM173 protein expression. We also found silencing of FKBP4 led to upregulation of TMEM173 at mRNA level in T47D and BT549 cells ( Figure 1I). Whereas overexpressed FKBP4 resulted in downregulation of TMEM173 at mRNA level in T47D and BT549 cells ( Figure 1J).
To further determine the effect of FKBP4 on TMEM173 transcriptional repression, we constructed TMEM173 promoter luciferase reporter plasmid. As shown in Figure 1K, L, siRNA speci cally targeting FKBP4 enhanced TMEM173 promoter activity in T47D and BT549 cells, and FKBP4-HA plasmid inhibited TMEM173 promoter activity in T47D and BT549 cells, therefore FKBP4 was rstly found to have an impact on regulating TMEM173 promoter.
We rstly used siRNA speci cally targeting FKBP4 in T47D and BT549 cells, and found it led to upregulation of NR3C1 at protein level ( Figure 2C). In addition, we transfected FKBP4-HA plasmid in T47D and BT549 cells, which resulted in downregulation of endogenous NR3C1 ( Figure 2D). These results clearly indicated that FKBP4 had a role in negatively regulating NR3C1 protein expression. Bioinformatics results also suggested a signi cant negative association between FKBP4 and NR3C1 both in luminal A and basal-like subtype of BC patients ( Figure S3A, B). Furthermore, we found naringenin could upregulate NR3C1 protein expression in T47D and BT549 cells ( Figure S4).

NR3C1 positively regulates TMEM173 at protein, mRNA and transcription level
We further con rmed the speci c interaction between NR3C1 and TMEM173 at the molecular level. We rstly used siRNA speci cally targeting NR3C1 in T47D and BT549 cells, which led to downregulation of TMEM173 at protein level ( Figure 2E). In addition, we transfected NR3C1-HA plasmid in T47D and BT549 cells, which resulted in upregulation of endogenous TMEM173 ( Figure 2F). These results clearly indicated that NR3C1 had a role in positively regulating TMEM173 protein expression. Given NR3C1 was demonstrated to regulate TMEM173 at protein level, we wondered whether it affected the mRNA level of TMEM173. We then found silencing of NR3C1 led to downregulation of TMEM173 at mRNA level in T47D and BT549 cells ( Figure 2G). Whereas overexpressed NR3C1 resulted in upregulation of TMEM173 at mRNA level in T47D and BT549 cells ( Figure 2H).
At the transcriptional level, siRNA speci cally targeting NR3C1 was found to decrease TMEM173 promoter activity in T47D and BT549 cells, while NR3C1-HA plasmid promoted TMEM173 promoter activity in T47D and BT549 cells ( Figure 2I, J), therefore NR3C1 was found to have a positive impact on regulating TMEM173 promoter.
Meanwhile, we veri ed a signi cant positive association between NR3C1 and TMEM173 both in luminal A and basal-like subtype of BC patients ( Figure S3C, D). Downregulated NR3C1 was signi cantly related to four molecular subtype patients than the normal group ( Figure S5), also strongly correlated with worse overall survival both in luminal A and basal-like subtype of BC patients ( Figure S6).
Besides, knockdown of NR3C1 partially prevented siRNA targeting FKBP4 to upregulate TMEM173 expression at protein, mRNA and transcription level in BT549 and T47D cells ( Figure 2K, L, M). Hence, our results implied that FKBP4 might downregulate TMEM173 by inhibiting NR3C1.

FKBP4 binds to NR3C1 and regulates nuclear translocation of NR3C1
As FKBP4 regulates its cellular targets via protein-protein interaction [41,42], we tested whether FKBP4 bound to NR3C1. We performed co-IP and western blot assays using BT549 and T47D cells, and results showed that FKBP4 and NR3C1 bound with each other (Figure 3A, B). We further observed that nuclear accumulations of NR3C1 were enhanced by siRNA targeting FKBP4 or inhibited by FKBP4-HA plasmid in BT549 and T47D cells by western blot analysis ( Figure 3C, D). Silencing FKBP4 mediated promotion of NR3C1 nuclear accumulations were also observed in BT549 and T47D cells by immuno uorescence microscopy ( Figure 3E, F).
These observations were consistent with the nding that FKBP4 transcriptionally inhibited TMEM173 through interacting with NR3C1.
All together, these results suggested that naringenin enhanced BC cell autophagy partially owing to FKBP4/NR3C1/TMEM173 axis.

FKBP4/NR3C1/TMEM173 signaling pathway involved in naringenin-restrained cell proliferation
Naringenin was well-known to inhibit cell proliferation [44], we doubted whether naringenin related FKBP4/NR3C1/TMEM173 axis was also involved in cell proliferation. Cell viability assay showed that silencing FKBP4 or overexpressing TMEM173 prevented cell proliferation of both T47D and BT549 cells at 72 h ( Figure 5A, B), while overexpressing FKBP4 or silencing TMEM173 promoted cell proliferation of both T47D and BT549 cells at 72 h ( Figure 5C, D). Similarly, we observed that silencing NR3C1 promoted cell proliferation and overexpressing NR3C1 prevented cell proliferation of both T47D and BT549 cells at 72 h ( Figure S8). Additionally, knockdown of TMEM173 signi cantly prevented naringenin-restrained cell proliferation of T47D and BT549 cells during 72 h ( Figure 5E, F), these ndings were further con rmed by cell photography ( Figure S9) and colony formation assay ( Figure S10). T47D cells treated with silenced TMEM173 were used to generate subcutaneous xenograft models in nude mice. Results showed that the volume and weight of tumors were signi cantly reduced in the naringenin treated group than in the control group, whereas those in the naringenin+shTMEM173 group were signi cantly greater than in the naringenin treated group ( Figure 5G, H, I). Immunohistochemical analysis showed that the staining intensity of FKBP4 was signi cantly lower in subcutaneous tumors in the naringenin treated group, and the expression of TMEM173 was higher than that in the control group ( Figure 5J).
Thus, in vivo and in vitro experiments indicated that naringenin inhibited BC proliferation partially owing to its function of increasing TMEM173 expression.

Exosomes secreted by naringenin treated breast cancer cells induced DC maturation
Exosomes secreted by BC cells had been reported as cell-to-cell mediators of oncogenic or anti-cancer information [45], and further regulated DC maturation [32], we wondered whether naringenin was also involved in exosome secretion among tumor environment. After 72 h naringenin treatment, exosomes were isolated from the supernatants of BC cells using a two-step procedure to achieve a high purity and their morphology veri ed by Nanoparticle tracking analysis ( Figure 6A). Next, puri ed exosomes derived from T47D and BT549 were further authenticated by the expression of exosome biomarkers (Alix, TSG101, CD81 and CD63) ( Figure 6B). The results of Nanoparticle tracking analysis showed that the isolated exosomes had an average size of 100 nm ( Figure 6C). Exosomes secreted by various cancer cells had been found to carry RNA and proteins, both western blot assays and RT-qPCR showed that the expression levels of NR3C1 and TMEM173 were higher in naringenin treated T47D exosomes (T47D-ex) and BT549 exosomes (BT549-ex) than in the control group, whereas FKBP4 had the opposite expression ( Figure 6D, We next tested the effect of exosomes secreted by naringenin treated BC cells on DC maturation. To this end, primary CD11c + DCs were isolated from the bone marrow of mice and cultured with exosomes for 72 h. DCs cultured with naringenin treated T47D-ex and BT549-ex showed signi cantly increased cell surface expression of co-stimulatory molecules CD40, CD80 and CD86 ( Figure 6F). We also observed the levels of IL-6, IL-12, TNF-α, IL-18, IL-1β and MCP-1, which re ected the maturation of DCs, were signi cantly upregulated by T47D-ex and BT549-ex treated with naringenin in the culture supernatant compared with those in the control group ( Figure 6G-L). Thus, T47D-ex and BT549-ex exhibited adjuvant properties of promoting DC maturation.

FKBP4/NR3C1/TMEM173 signaling pathway involved in naringenin-induced TIDC maturation
To con rm FKBP4/NR3C1/TMEM173 axis was also regulated in DC, we co-cultured BC cells and bone marrow cells of mice with GMCSF+IL4 or naringenin, simulated as TIDC ( Figure 7A). After GMCSF+IL4 treatment for 72 h, we found that FKBP4 was gradually decreased while NR3C1 and TMEM173 were gradually increased during TIDC differentiation and maturation at both protein and mRNA level ( Figure 7B, C). After naringenin treatment for 72 h, we found that FKBP4 was gradually decreased while NR3C1 and TMEM173 were gradually increased according to naringenin concentration at both protein and mRNA level ( Figure 7D, E). Flow cytometry analysis results showed that the positive expression percentages of CD11C and CD86 of TIDC were signi cantly increased in the naringenin group in contrast to those in the control group ( Figure 7F). To demonstrate that TMEM173 truly affected the maturation and function of TIDC, we observed the levels of IL-6, IL-12, TNF-α, IL-18, IL-1β and MCP-1 were signi cantly upregulated by naringenin stimulation in the TIDC culture supernatant compared with those in the control group, opposite results were observed in the groups receiving anti-TMEM173 stimulation ( Figure 7G-L). These data suggested that FKBP4/NR3C1/TMEM173 signaling pathway involved in naringenin-induced TIDC maturation.
Taken together, we demonstrated that naringenin mediated pro-autophagy, anti-proliferation of BC cells and pro-TIDC maturation in a new-found FKBP4/NR3C1/TMEM173 dependent way (Figure 8).
Recently, endogenous host-derived regulators of TMEM173 have become the hotspot on related researches. For instance, an in ammasome protein called NOD-like receptor family CARD domain containing 3 (NLRC3) blocked the direct tra cking of TMEM173 to punctuated endoplasmic-associated puncta region by direct binding to TMEM173 [46]. The autophagy protein P62 negatively regulated the TMEM173 signaling by initiating the ubiquitination of TMEM173 to autophagosome [47]. As an ER membrane-bound E3-ubiquitin ligase, the RING nger protein 5 degraded TMEM173 by mediating its K48linked polyubiquitination [48]. Unlike the above-mentioned factors inhibiting TMEM173 at protein level, the current study suggested FKBP4/NR3C1 axis might be a novel endogenous negative regulator of TMEM173 by altering its transcriptional activity in both BC cells and TIDCs. Thus, FKBP4 and NR3C1 could serve as notable therapeutic molecules against TMEM173-dependent tumorigenesis, autoin ammation and autoimmunity in the future.
FK506-binding protein family in human genomes has included 18 FKBPs up to date, which could target on various pathways in embryonic development, stress response, cardiac function, cancer tumorigenesis and neuronal function [49]. In colorectal cancer, silencing FKBP3 has been found to attenuate oxaliplatin resistance by regulation of the phosphatase and tensin homolog/AKT axis [50]. In Alzheimer's disease, FKBP12 and amyloid precursor protein interplay has suspected to affect Aβ peptides expression [51]. Although FKBP4 has been demonstrated to connect mammalian target of naringenin complex 2 and phosphoinositide-3-kinase to enhance cell proliferation of BC, for the rst time, we found that FKBP4 played a carcinogenic role by downregulating TMEM173 in BC cells and co-cultured DCs.
Currently, there is no research of regulation mechanisms on NR3C1 and cGAS-STING pathway, but only a few studies suggest that NR3C1 is involved in the innate immune response [52,53]. In our study, we demonstrated FKBP4 transcriptionally downregulated TMEM173 through binding to NR3C1, thus inhibiting nuclear translocation of NR3C1, but this effect was found relatively weak because shNR3C1 could not completely inhibit FKBP4 from regulating TMEM173 according to results of Fig. 2K. Therefore, we speculated that some of the predicted TFs were also involved in FKBP4/TMEM173 pathway, subsequent mechanism studies need to be further improved to verify our conjecture.
As for the interaction of FKBP and autophagy, FKBP5 has been reported to change phosphorylation of Beclin1 by binding to Beclin1, thus triggering autophagic pathways [54]. FKBP8 was found to recruit lipidated LC3A to induce mitochondrial autophagy via N-terminal LC3-interacting region motif of FKBP8 [55]. While the regulation of autophagy by FKBP4 in tumor cells has not been reported yet. Meanwhile, TMEM173 could activate autophagy through inducing LC3B lipidation, which was independent of TANK binding kinase 1 activation and interferon induction [56]. In current study, we rstly demonstrated that inhibiting FKBP4 could promote autophagy by increasing TMEM173 expression of BC cells.
TMEM173 expression has been reported suppressed or lost in majority of cancers, especially in BC [57][58][59], which suggests that during BC progression, downregulation of the proteins involved in innate immune response may be helpful for evading innate immune response pathways to facilitate tumor growth.
Although hypermethylation of the promoter regions in TMEM173 has been revealed in colorectal cancer [15], little is known about the mechanism of decreased TMEM173 expression in BC. Our results imply FKBP4 could induce TMEM173 transcriptional suppression via NR3C1, it would be interesting to further study the correlation between TMEM173 and other FKBPs involved in innate immune response and relevance during breast tumorigenesis.
Demaria et al. demonstrated that IFN-stimulatory dsDNA from irradiated BC cells, which are phagocytosed by DCs via stimulating cGAS/TMEM173 pathway [32]. Thus, it is likely that different methods mediate the transfer of other cargos to DCs through TMEM173 related signalings, e.g., our study rstly found that exosomes secreted by naringenin treated BC cells induced DC maturation on TMEM173 dependent way.
Further experimentation will be required to determine the relative contribution of these different mechanisms of tumor DNA delivery to DCs.
Recently, a growing body of evidence has demonstrated that the pharmacological agents, e.g.,

Conclusions
In summary, we demonstrated that FKBP4 transcriptionally downregulated TMEM173 through inhibiting nuclear translocation of NR3C1, naringenin promoted autophagy, suppressed proliferation in luminal A and basal-like subtype of BC cells and enhanced TIDC maturation via new-found FKBP4/NR3C1/TMEM173 axis. We proposed that FKBP4/NR3C1/TMEM173 signaling pathway served as a potential immunological therapeutic target for the treatment of BC patients.

AVAILABILITY OF DATA AND MATERIAL
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

COMPETING INTERESTS
5. Zhong J, Yu R, Zhou Q, Liu P, Liu Z, Bian Y: Naringenin prevents TNF-α-induced gut-vascular barrier disruption associated with inhibiting the NF-κB-mediated MLCK/p-MLC and NLRP3 pathways.  FKBP4 binds to NR3C1 and FKBP4 inhibition triggers nuclear translocation of NR3C1. a, b co-IP and western blot assays using antibodies as indicated for binding between endogenous FKBP4 and NR3C1 in T47D and BT549 cells. c, d T47D and BT549 cells were transfected with siRNAs or plasmids. Cells were harvested, and the nuclear and cytoplasmic fractions were separated. Proteins were analyzed by immunoblotting using anti-NR3C1, anti-FKBP4, anti-GAPDH, or anti-histone antibody (n=3 independent biological replicates). e, f T47D and BT549 cells were transfected with siRNAs. Immuno uorescent staining was carried out using anti-NR3C1 and anti-FKBP4. DAPI staining was performed to show the nuclei (n=3 independent biological replicates). The arrowheads show the NR3C1 nuclear localization under FKBP4 inhibition. Scale bar=20μm.  replicates). d Bar chart showed silencing TMEM173 resulted in increased cell viability of T47D and BT549 cells at 72 h (n=3 independent biological replicates). e, f Line chart showed knockdown of TMEM173 by shRNA attenuated naringenin-restrained cell proliferation in T47D and BT549 cells treated by 100 nM naringenin, numeric representation at each dot is cell viability fold change of 0nM and 100 nM naringenin treatment (n=3 independent biological replicates). g, h, i The macroscopic appearance, volume and weight of subcutaneous tumors in mice (n=3/group) transplanted with T47D cells treated with shControl, shControl+Naringenin and shTMEM173+Naringenin. j The expression of FKBP4 and TMEM173 in mice transplanted with T47D cells treated with shControl, shControl+Naringenin and shTMEM173+Naringenin were determined by using immunohistochemistry. *p<0.05, **p<0.01, ***p<0.001.

Figure 6
Identi ed breast cancer exosomes promote dendritic cell maturation. a Schema of breast cancer exosomes isolation method. b Western blot analysis of exosome biomarkers (Alix, TSG101, CD81 and CD63). c Particle size distribution of exosomes measured by Nanoparticle tracking analysis. d, e The expression of NR3C1, FKBP4 and TMEM173 in T47D-ex and BT549-ex were examined by using western blot analysis and RT-qPCR. f CD11c+ DCs were cultured for 72 h with T47D-ex and BT549-ex (30 μg) and analyzed for expression of co-stimulatory molecules by ow cytometry analysis. Mean uorescence intensity (MFI)±s.e.m. of samples in each group (n=3 independent biological replicates). MFI was calculated after subtracting background. g-l The level of cytokines IL-6, IL-12, TNF-α, IL-18, IL-1β and MCP-1 secretion in DC culture supernatants were measured by ELISA. *p<0.05, **p<0.01, ***p<0.001.

Figure 7
FKBP4/NR3C1/TMEM173 signaling pathway involved in naringenin-induced TIDC maturation. a Schema of mice bone marrow cells co-cultured with BC cells. b Western blot analysis of NR3C1, FKBP4 and TMEM173 protein expression in DC co-cultured with BC cells for 3 days. c RT-qPCR analysis of NR3C1, FKBP4 and TMEM173 mRNA expression in DC co-cultured with BC cells for 3 days. d, e The expression of NR3C1, FKBP4 and TMEM173 in TIDC on different concentration of naringenin were examined by using western blot analysis and RT-qPCR. f TIDC were treated with different concentration of naringenin for 72 h and analyzed for maturation by ow cytometry analysis. g-l The level of cytokines IL-6, IL-12, TNF-α, IL-18, IL-1β and MCP-1 secretion in TIDC culture supernatants were measured by ELISA. *p<0.05, **p<0.01, ***p<0.001.

Figure 8
Model of naringenin-mediated cell proliferation inhibition and cytoprotective autophagy of BC cells and TIDC maturation promotion through FKBP4/NR3C1/TMEM173 signaling pathway. Naringenin inhibits FKBP4, thus releasing and inducing NR3C1 to bind to the TMEM173 gene promoter, which triggers upregulation of TMEM173 mRNA and protein level, leading to proliferation inhibition and autophagy promotion of BC cells, as well as maturation promotion of TIDC. Among tumor microenvironment, naringenin could regulate the expression of FKBP4, NR3C1 and TMEM173 carried by BC exosomes, thus enhancing maturation and antigen presenting function of TIDC.

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