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, 38–40].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 first 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 . However, Ballou et al.  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 specific . 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 . For instance, our previous study demonstrated that 5-HT1B and 5-HT1D receptors contribute in pancreatic cancer cells growth, invasion, and progression . Also, in other our study showed that 5-HT1B receptor promotes uterine leiomyoma cell survival and proliferation . Liang et al.  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 , non-small cell lung cancer , hepatocellular carcinoma , and glioblastoma . Although 5-HT7 receptor is expressed in the involution of the mammary gland  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 significantly shorther patient survival, suggesting that 5-HT7 is a clinically significant prognostic factor that contributes to an oncogenic signaling in breast cancer. Furthermore, 5-HT treatment dose-dependently increased 5-HT7 receptor and FOXM1 expression and cell proliferation in TNBC cells. Also, we showed that inhibition of 5-HT7 receptor by specific siRNA and metergoline, a 5-HT7 receptor antagonist, significantly 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 significantly decreased, when 5-HT7 receptor is blocked by pharmacologically using 5-HT7 receptor antagonist or genetically using 5-HT7 receptor specific siRNA. We demostrated that 5-HT/HT7 axis promotes FOXM1 oncogenic signaling to induce TNBC cell proliferation and survival as inhibition of FOXM1 significantly 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  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 . Overall, all of these findings 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 specific antagonist for the only 5-HT7 receptor . 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 first 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 significant 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  suggesting that HT7 targeted therapeutics may be more selective towards TNBC cells.
In conclusion, our findings 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.