Serotonin 5-HT7 Receptor Is A Biomarker Poor Prognostic Factor And Induces Proliferation of Triple Negative Breast Cancer Cells Through FOXM1

Purpose: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer and associated with poor prognosis and shorter survival due to signicant genetic heterogeneity, drug resistance and lack of effective targeted therapeutics. Therefore, novel molecular targets and therapeutic strategies are needed to improve patient survival. Serotonin (5-hydroxytriptamine, 5-HT) has been shown to induce growth stimulatory effects in breast cancer. However, the molecular mechanisms by which 5-HT exerts its oncogenic effects in TNBC still are not well understood. Methods: Normal breast epithelium (MCF10A) and two TNBC cells (MDA-MB-231, BT-546) and MCF-7 cells (ER+) were used to investigate effects of 5-HT7 receptor. Small interfering RNA (siRNA)-based knockdown and metergoline (5-HT7 antagonist) were used to inhibit the activity of 5-HT7. Cell proliferation and colony formation were evaluated using MTS cell viability and colony formation assays, respectively. Western blotting was used to investigate 5-HT7, FOXM1 and its downstream targets protein expressions. Results: We demonstrated that 5-HT induces cell proliferation of TNBC cells and expression of 5-HT7 receptor and FOXM1 oncogenic transcription factor. We found that expression of 5-HT7 receptor is upregulated in TNBC cells and higher 5-HT7 expression is associated with poor patient prognosis and shorter patient survival. Genetic and pharmacological inhibition of 5-HT7 by siRNA and metergoline, respectively, suppressed TNBC cell proliferation and FOXM1 and its downstream mediators, including eEF2-Kinase (eEF2K) and cyclin-D1. Conclusion: Our ndings suggest for the rst time that the 5-HT7 receptor promotes FOXM1, eEF2K and cyclin D1 signaling to support TNBC cell proliferation, thus inhibition of 5-HT7/FOXM1 signaling may be used as a potential therapeutic strategy for targeting TNBC.


Introduction
Triple negative breast cancer (TNBC) accounts for approximately 10-15% of all breast cancers and is de ned by the lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 expression (HER2).TNBC is characterized by a highly invasive phenotype, early metastasis, poor prognosis, and limited response to conventional chemotherapies and lack of effective targeted treatment options [1]. Although, in 2020 a new targeted treatment strategy was approved by the FDA for patients with metastatic TNBC, the overall response rate was low with a median duration of response of 7.7 months [2][3][4]. Therefore, identi cation of novel molecular targets and development effective therapeutic strategies are urgently needed for treating TNBC.
Serotonin (5-hydroxytryptamine; 5-HT) is a biogenic monoamine neurotransmitter and also, acts as a regulatory hormone involved in controlling various physiological processes [5]. Although 5-HT is mostly known as a neurotransmitter produced by tryptophan hydroxylase 2 (TPH2) in the central nervous system, it is also synthesized by epithelial cells in mammary gland through tryptophan hydroxylase 1 (TPH1) and acts in regulation of epithelial homeostasis in breast cancers [6]. The alteration of 5-HT and 5-HT receptor expression patterns leads to dysregulation of epithelial homeostasis which has been associated with breast cancer initiation, progression and tumorigenesis [6][7][8][9][10]. Furthermore, it is shown that 5-HT has been linked to the development of other cancers [11][12]. These studies have suggested that 5-HT acts as a mitogenic signal for cancer cells and, contributes to cell proliferation in various cancers including breast cancer [5,9]. Moreover, it is shown that 5-HT activates oncogenic signal pathway such as JAK/STAT3/ERK [137], MAPK [7] and Akt/mTOR [10] in breast cancer cells.
Furthermore, it was reported that free 5-HT levels in plasma may be used as a marker in the early detection of breast cancer, since 5-HT is produced in high levels in breast cancer cells [13].
However, the exact molecular mechanism by which 5-HT7 receptor exerts oncogenic signaling in breast cancer cells are not understood.
Forkhead box M1 (FOXM1) is a multi-functional transcription factor, and an important important molecular driver of TNBC metastasis and tumorigenesis [21,22]. We recently showed that FOXM1 transcriptionally regulates expression of eukaryotic elongation factor-2 kinase (eEF2K) in TNBC cells, promoting TNBC tumor growth and metastasis [21], indicating that FOXM1 represents an important molecular target in TNBC [21,22].In the current study, we aimed to investigate the molecular mechanisms of 5-HT7 receptor-mediated effects in TNBC cells. Given the oncogenic effects of 5-HT in breast cancer cells, we hypothesized that 5-HT signaling may contribute to oncogenic progression of TNBC via FOXM1.
We found that 5-HT induced TNBC cell proliferation and expression of 5-HT7 and FOXM1. We also found that metergoline, a high-a nity 5-HT7 receptor antagonist [23], and 5-HT7 speci c siRNA reduced TNBC cell proliferation and colony formation through inhibition FOXM1 and its downstream targets, including eEF2K and cyclin-D1 in TNBC cells. Overall, our ndings suggest for the rst time that 5-HT/5-HT7 signaling contributes to cell proliferation FOXM1/eEF2K/cyclin-D1 axis, representing a potential molecular target to control TNBC growth.
Transfection with siRNA Small interfering RNAs (siRNAs) targeting Serotonin-receptor 7 (5-HT7) and Forkhead box M1 (FOXM1) genes and non-silencing control siRNA were purchased from Sigma-Aldrich. Exponentially growing cells were plated 24h before transfection and transfected with 5-HT7 siRNA, two different FOXM1 siRNAs or control siRNA at a nal concentration of 50 nM and 100 nM for 72h, using HiPerFect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer's protocol. Non-silencing control siRNAtransfected cells were used as negative controls. After treatment, the cells were harvested and processed for further analysis [21,25,26].

Colony formation assays
To detect the effect of metergoline on cell proliferation and colony formation of breast cancer, we performed clonogenic assay as described previously [25]. TNBC cells were seeded in 6-well plates (1.5 × 10 3 cells/ well); treated with increasing doses of metergoline (1, 5, 10, 15, 20, 25 and 30 µM) for and incubated at 37°C for 2 weeks to form colonies. Furthermore, cells were seeded in 6-well plates (1.5 x 10 3 cells/well) transfected with a non-silencing control siRNA or 5-HT7 siRNA (50 nM and 100 nM) and FOXM1 siRNAs (50 nM), and grown for 2 weeks. The cells were washed with PBS and stained with crystal violet, and visible colonies were counted [21,26].

Western Blot analysis
For western blot analysis, cells were seeded in T-25 culture asks and treated with 5-HT or metergoline at indicated concentrations and time points. MDA-MB-231 cells were treated with 30-35 µM metergoline for 48h and 20 µM metergoline for 72 h. BT-549 cells were treated with 20 and 30 µM metergoline for 48h and 10 µM metergoline for 72 h. Furthermore, both MDA-MB-231 and BT-549 cells were transfected with siRNAs (50 nM and 100 nM) for 72 h. Then, the cells were collected, washed twice in ice-cold phosphatebuffered saline (PBS) and lysed in a lysis buffer at 4°C. The protein concentrations were measured with a protein assay kit (DC kit; Bio-Rad, Hercules, CA). A total of 40 µg of protein from each sample was separated by Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis with a 4-20% gradient and transferred to polyvinylidenedi uoride membranes. The membranes were blocked with a blocking buffer (0.1 Triton X-100 with 5% dry milk in Tris-buffered saline-Tween 20 (TBS-T) for 60 min. After being washed with TBS-T, the membranes were probed with the following primary antibodies: FOXM1 (Cell Signaling), p-EF2 (Cell Signaling), EF2 (Cell Signaling), EF2K (Proteintech), 5-HT7 (YLBiont), Cyclin-D1 (Proteintech) and β-actin (Proteintech). After being washed with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Bio-Rad, #170-6515) or anti-mouse secondary antibody ((Bio-Rad). β-actin was used as a loading control. All antibodies were diluted in TBS-T containing 5% dry milk. Chemiluminescence detection was performed with Clarity Western ECL Substrate (Biorad) and the blots were visualized with a ChemiDoc MP Imaging System (Biorad) and quanti ed with a densitometer using the ChemiDoc MP Imager application program (Biorad) [21,[24][25][26]].

Statistical analysis
All experiments were conducted at least in triplicate, and the results were summarized as means with standard deviations. Statistical signi cance was determined using the Student t test. p values less than 0.05 were considered statistically signi cant. GraphPad Prism program was used to evaluate the data and plot the graphs.

5-HT induces cell proliferation of TNBC cells
To determine the effects of 5-HT on TNBC (MDA-MB-231 and BT-549) and ER+ (MCF-7) cells proliferation and viability, we performed MTS assay after 24 h and 48 h treatment with increasing doses of 5-HT (1, 2 and 4 µM). MTS assay revealed that TNBC cells were more sensitive to 5-HT and showed a signi cant increase in the number of viable cells at all concentrations and at different time points compared to untreated (NT) and EtOH-treated control cells (Fig. 1, a-d). However, while no signi cant change was observed in number of MCF-7 cells treated with increasing concentration of 5-HT for 24h (Fig. 1, e), we found a signi cant increase in the number of cell viability at all concentrations after 48 h (Fig. 1, f). These results indicated that TNBC cells more responsive to 5-HT induced cell proliferation compared to ER+ MCF7 cells which were less sensitive to 5-HT-induced effects.
5-HT7 receptor is overexpressed in breast cancer cells and its expression is associated with shortened overal survival in BC patients 5-HT7 receptor has been shown to play a role in mediating 5-HT-induced mitogenic effects in TNBC [8,19]. Therefore, we investigated the clinical signi cance of 5-HT7 expression in breast cancer patients, and we analyzed the NCI-The Cancer Genome Atlas (TCGA) breast cancer database. Kaplan-Meier survival analysis demonstrated that the overall survival rate was considerably shorter in patient tumors with high 5-HT7 gene expression (n=122 patients) compared to those with low 5-HT7 expression (n=282) (p=0.022) ( Fig. 2, a).
Next, we examined 5-HT7 receptor expression in two TNBC (MDA-MB-231 and BT-549) and one ER+ (MCF-7) breast cancer cell line, and found that protein expression level of 5-HT7 receptor in TNBC were much higher than MCF-7 cells and MCF10A normal epithelial cells (Fig. 2, b, c).

5-HT modulates 5-HT7 and FOXM1 signaling in TNBC cells
Although recent studies showed that 5-HT7 is also involved in proliferation of MDA-MB-231 cells [8,19], the mechanisms by which it is responsible are still unclear. We and others have previously shown that FOXM1 is upregulated in TNBC cells and required for cell proliferation and survival in TNBC [21,27]. Also, according to analysis of The Cancer Genome Atlas breast cancer data base, we have reported that FOXM1 expression is associated with poor prognosis and shorter patient survival in breast cancer [25]. Therefore, we hypothessized that 5-HT/ 5-HT7 receptor signaling may induce FOXM1 to promote cell proliferation in TNBC cells. To con rm our hypothesis, we rst investigated expression pattern of FOXM1 in highly aggressive and metastatic TNBC cells and non-invasive MCF7. We found that FOXM1 expression displays a similar expression pattern to the 5-HT7 expression pattern in breast cancer cells ( Fig. 2, b-d). We also found that 5-HT treatment (2 µM and 4 µM for 48 h) led to increase 5-HT7 and FOXM1 expression by western blot analysis in MDA-MB-231 (Fig. 2, e-g) and BT-549 (Fig. 2, h-j) cells compared to the control cells.These results indicated that 5-HT/5-HT7 signaling may modulate FOXM1 expression in TNBC cells.

Inhibition of 5-HT7 by an antagonist inhibitor suppresses cell proliferation and FOXM1 signaling in TNBC cells
To investigate effects of 5-HT7 on TNBC cells proliferation, we rst treated cells with a 5-HT7 antagonist metergoline [23,28,29] with increasing concentration (1, 10, 20, 30 and 35 µM) for 48h and 72h. The MTS analysis revealed that metergoline treatment signi cantly reduced cell proliferation of MDA-MB-231 (Fig. 3, a, b) and BT-549 cells (Fig. 3, c, d) compared to untreated (NT) and EtOH-treated control cells. We then investigated the effects of metergoline on colony formation of MDA-MB-231 and BT-549 cells. Metergoline treatment (1 to 30 µM) led to a signi cant reduction in the number of colonies at doses starting from 1µM both MDA-MB-231 and BT-549 cells compared to NT and EtOH-treated cells (Fig. 3, eh). BT-549 cells were more sensitive to metergoline treatment that completely inhibited cell proliferation starting from 15 µM in BT-549 cells while the similar affects were observed in MDA-MB-231 cells at doses starting from 25 µM (Fig. 3, e-h). More importantly, we found that metergoline treatment led to a marked reduction in the expression FOXM1 protein in TNBC cells (Fig. 4, a-c, e-g). Moreover, both 5-HT7 receptor and FOXM1 expressions were inhibited in response to the treatment with metergoline (Fig. 4, a-h). These ndings suggested that 5-HT7 may promote cell proliferation by upregulating FOXM1 expression in TNBC cells.

Knockdown of 5-HT7 suppresses cell proliferation and FOXM1 expression in TNBC cells
Although metergoline has a high a nity antagonist for the 5-HT7 receptor [23,28,30], it may also act as an antagonist for 5-HT1 and 5-HT2 serotonin receptors [31][32][33]. Therefore, to con rm the direct involvement of 5-HT7 in TNBC cell proliferation and FOXM1 expression, we knocked down 5-HT7 using speci c siRNA targeting its mRNA in TNBC cells. To this end, the TNBC cells were transfected with 50 or 100 nM 5-HT7-siRNA, and about two weeks later we examined colony formation. We found that knockdown of 5-HT7 resulted in a marked reduction of cell proliferation and colony formation in MDA-MB-231 (Fig. 5, a, b) and BT-549 cells (Fig. 5, c, d) compared to the control siRNA transfected cells. BT-549 cells were more sensitive to 5-HT7 inhibition by siRNA and had much more reduced number of colonies compared to MDA-MB-231 cells treated with HT7 siRNA. Overall, these results indicated that 5-HT7 is involved in survival, proliferation of TNBC cells.

Knockdown of 5-HT7 inhibited downstream targets of FOXM1/eEF2K signaling in TNBC cells
We have previously demonstrated that eEF2K promotes cell proliferation and tumor growth and progression of TNBC [34][35][36]. We have also reported that FOXM1 transcriptionaly regulates expression of eEF2K and FOXM1 inhibition leads to downregulation of eEF2K and supresses cell proliferation and tumor growth in TNBC models [21]. Therefore, to elucidate the molecular mechanism of 5-HT7-induced cell proliferation, we also investigated whether knocked down of 5-HT7 leads to inhibition downstream targets of FOXM1 such as eEF2K, p-EF2 and cyclin D1. As expected, knockdown of 5-HT7 receptor by siRNA decreased the eEF2K expression and phosphorylation of EF2 (p-EF2) at Thr56 in MDA-MB-231 (Fig. 5, e, h, i) and BT-549 cells (Fig. 5, l, o, p) compared to control cells. Interestingly, expression level of eEF2 were signi cantly decreased after transfection with 5-HT7-siRNA in BT-549 cells (Fig. 5, l, r). Cyclin-D1 promotes the cell cycle entry by inducing G1/S phase transition, also, it is downstream target of FOXM1 [21]. We found that Cyclin-D1 expression was signi cantly suppressed in MDA-MB-231 ( Fig. 5, e, k) and BT-549 (Fig. 5, l, s) cells transfected with 5-HT7-siRNA. Overall, our results indicated that 5-HT7 receptor regulates the FOXM1/eEF2K/cyclin-D axis, promoting cell proliferation in TNBC cells.

Inhibition of FOXM1 impairs proliferation of TNBC cells
To further demonstrate that the mechanism by which 5-HT7 regulates cell proliferation through FOXM1 expression, we investigated the effects of FOXM1 knockdown on cell proliferation of TNBC cells. Knockdown of FOXM1 by siRNA in MDA-MB-231 and BT-549 cells resulted in a marked reduction of colony formation compared with control siRNA-transfected MDA-MB-231 (Fig. 5, t-u) and BT-549 cells (Fig. 5, v-y). Overrall, these ndings indicate that 5-HT7-induced TNBC cell proliferation is mediated by FOXM1expression.

Discussion
Recent studies have demonstrated a potential stimulatory effect of 5-HT in promoting cell proliferation, angiogenesis, invasion, migration and metastasis in various of cancer types [8,11,21,37]. Interestingly. 5-HT is produced by several types of tumor cells, including breast cancer through upregulation of the 5-HT producing enzyme TPH1 [8,[38][39][40].Therefore, 5-HT mediated signaling is proposed as a potential therapeutic target. However, molecular mechanisms by which 5-HT acts as a growth factor and promotes mitogenic effects is not well understood [7,9,14]. Results of the current study demostrated for the rst time that 5-HT induces oncogenic FOXM1/eEF2K and cylin D1 signaling axis through HT7 receptor in TNBC cells.

5-HT has been shown to exert a mitogenic effect in various cancer cells.
It is demonstrated that hepatocellular cancer cells exhibited increase in cell viability dose-dependent manner after treated with 5-HT, and predominantly promotes cell proliferation of hepatocellular cancer cells [11]. However, Ballou et al. [7] did not observe 5-HT mediated an increase in cell proliferation of breast cancer, but found that serotonin stimulation enhances activating phosphorylation of key mitogenic regulators such as Akt2, CREB, MAPK. In another study showed that 5-HT stimulates cell proliferation, even, mitogenic effect of 5-HT is TNBC cells speci c [8]. Similar results were reported by other authors, and where authors, indicated that an effect mitogenic of 5-HT had been for breast cancer cells [14,41]. In presented study showed that 5-HT stimulated cell viability and proliferation in TNBC. As a results, both our study and other studies clearly indicate that 5-HT is signaling proliferative positive support to TNBC cells, by increasing the rate of cell viability and proliferation.
It is known that 5-HT signaling through various 5-HT receptors regulates the survival and proliferation of cancer cells [9]. For instance, our previous study demonstrated that 5-HT1B and 5-HT1D receptors contribute in pancreatic cancer cells growth, invasion, and progression [42]. Also, in other our study showed that 5-HT1B receptor promotes uterine leiomyoma cell survival and proliferation [43]. Liang et al. [44] reported that serotonin mediates the proliferation of hepatocellular carcinoma cells though 5-HTB2 receptor. Similarly, oncogenic effects of 5-HT on breast cancer cells are mediated through several receptors such as 5-HT1, 5-HT2, 5-HT2A and 5-HT3A [5,7,39,41,45]. Eventually, all of studies reportes that some 5-HT receptors subtypes are considered potential targets for treatment of several cancers [8,9].
Although functions of many 5-HT receptors subtypes have been studied in various cancer types, studies regarding the role of 5-HT7 receptor in TNBC are very limited. Previous studies indicated that 5-HT7 receptor is associated with proliferation in prostate cancer [46], non-small cell lung cancer [47], hepatocellular carcinoma [11], and glioblastoma [48]. Although 5-HT7 receptor is expressed in the involution of the mammary gland [20] and breast cancer cells [7,8,14,19,20], there are only several studies investigating function of the 5-HT7 receptor in breast cancer cells. These studies reported that 5-HT induces MDA-MB-231 TNBC cell proliferation and invasion [14,19,20]. Our current study demonstrated that 5-HT7 receptor is overexpressed in TNBC and associated with poor patient prognosis and signi cantly shorther patient survival, suggesting that 5-HT7 is a clinically signi cant prognostic factor that contributes to an oncogenic signaling in breast cancer. Furthermore, 5-HT treatment dosedependently increased 5-HT7 receptor and FOXM1 expression and cell proliferation in TNBC cells. Also, we showed that inhibition of 5-HT7 receptor by speci c siRNA and metergoline, a 5-HT7 receptor antagonist, signi cantly suppressed TNBC cell proliferation, suggesting that 5-HT7 is promoting oncogenic signaling.
We have previously demonstrated that FOXM1 is overexpressed in TNBC cells and higher expression of FOXM1 is associated with shorter survival and worse prognosis of patients [21,25]. In addition, FOXM1 regulates multiple oncogenic signaling pathways that are involved in cell proliferation, migration, invasion, and autophagy in TNBC [21,26,49]. In presented study showed that FOXM1 expression correlates with levels of HT7, which also plays a critical role in cell proliferation and cell cycle progression in TNBC cells [21,27].
Intracellular signaling pathways linked to mitogenic action of 5-HT are still unclear in breast cancer cells [9,14]. Therefore, we investigated the downstream signaling molecules responsible for transmitting 5-HT/5-HT7 receptor signaling that leads to TNBC cell proliferation. We found a dose-depended induction in the FOXM1 expression levels by 5-HT. Moreover, we found that expression level of FOXM1 was signi cantly decreased, when 5-HT7 receptor is blocked by pharmacologically using 5-HT7 receptor antagonist or genetically using 5-HT7 receptor speci c siRNA. We demostrated that 5-HT/HT7 axis promotes FOXM1 oncogenic signaling to induce TNBC cell proliferation and survival as inhibition of FOXM1 signi cantly suppressed cell proliferation in TNBC cell. We and others have previously shown that oncogenic transcription factor FOXM1 promotes TNBC proliferation, migration and invasion and tumor growth in mice models [21,27]. Furthermore, we demonstrated that knockdown of 5-HT7 receptor by siRNA resulted in decreased expression level of eEF2K, which is a transcriptional target of FOXM1 [21] and one of the important oncogenic signaling pathways that promotes cell proliferation, invasion and tumorigenesis in TNBC [34,35,36]. Furthermore, we found that 5-HT7 knockdown reduced expression of cyclin-D1, which plays a key role in G1 phase, G1/S transition and oncogenesis, and recently shown to be regulated by FOXM1 and eEF2K in TNBC cells [21]. Overall, all of these ndings suggest that 5-HT/ 5-HT7 receptor signaling may contribute to proliferation and survival of TNBC cells by regulating FOXM1 and its downstream mediators, including eEF2K and cyclin-D1. These results also suggest that 5-HT/ 5-HT7 signaling may be a promising therapeutic target for TNBC. 5-HT7 antagonists such as metergoline have been considered for the treatment of breast cancer, but there is no speci c antagonist for the only 5-HT7 receptor [15]. Metergoline irreversibly blocks the 5-HT7 receptors and provides a profound inactivation of the 5-HT7 receptor [23,28,29]. In presented study, we showed that metergoline treatment led to a marked inhibition of 5-HT7 receptor expression in TNBC cells.
Also, we found for the rst time that the mitogenic effect of 5-HT7 receptor was blocked by metergoline treatment, which suppressed FOXM1 expression and downstream signaling, including eEF2K and cyclin D1 in TNBC cells. As expected, inhibition of FOXM1 with metergoline leads to signi cant reduced of cell proliferation and survival in TNBC cells. Therefore, in this study, we suggest that metergoline may be as a potential anti-cancer agent that suppresses TNBC progression, by inhibiting FOXM1 expression, and thus, suppressing survival and proliferation of TNBC cells promoted by 5-HT. Considering the fact that expression level of 5-HT7 was lower in MCF10A normal breast epithelial cells and 5-HT does not stimulate the cell proliferation [8] suggesting that HT7 targeted therapeutics may be more selective towards TNBC cells.
In conclusion, our ndings demonstrated that 5-HT/ 5-HT7 receptor signaling plays an important role in TNBC cell proliferation by regulating FOXM1/eEF2K/Cyclin-D1 axis in TNBC cells. Thus inhibition of 5-HT7/FOXM1 signaling may be used as a potential therapeutic approach for controlling TNBC. Further studies are needed to explore this therapeutic strategy in vivo TNBC tumor models to determine whether this therapeutic approach is valid for controlling TNBC tumor growth and progression.  Expressions of 5-HT7 and FOXM1 proteins were determined by Western blot. Protein expression intensities were evaluated by densitometric analysis in MDA-MB-231 (j, k) and BT-549 cells (m, n) (ns: non-signi cant, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001). All experiments were independently repeated at least twice.

Figure 3
Metergoline treatment inhibits cell proliferation and colony formation in TNBC cells Cells were treated with increasing concentration of metergoline, and cell proliferation was evaluated after 48 h and 72 h by MTS assay (a-d). Cells were treated with increasing concentration of metergoline and evaluated for colony formation in MDA-MB-231 (e, f) and BT-549 (g, h). (ns: non-signi cant, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001). All experiments were independently repeated at least twice.