3. 1. 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 significant 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 significant change was observed in number of MCF-7 cells treated with increasing concentration of 5-HT for 24h (Fig. 1, e), we found a significant 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.
3. 2. 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 significance 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).
3. 3. 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 . Therefore, we hypothessized that 5-HT/ 5-HT7 receptor signaling may induce FOXM1 to promote cell proliferation in TNBC cells. To confirm our hypothesis, we first 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.
3. 4. 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 first 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 significantly 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 significant 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, e-h). 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 findings suggested that 5-HT7 may promote cell proliferation by upregulating FOXM1 expression in TNBC cells.
3. 5. Knockdown of 5-HT7 suppresses cell proliferation and FOXM1 expression in TNBC cells
Although metergoline has a high affinity antagonist for the 5-HT7 receptor [23, 28, 30], it may also act as an antagonist for 5- HT1 and 5-HT2 serotonin receptors [31-33]. Therefore, to confirm the direct involvement of 5-HT7 in TNBC cell proliferation and FOXM1 expression, we knocked down 5-HT7 using specific 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.
Next, we evaluated expressions of FOXM1 and 5-HT7 proteins in 5-HT7 siRNA transfected TNBC cells by Western blot. Knockdown of 5-HT7 by siRNA significantly inhibited expression of FOXM1 and 5-HT7 receptor in both MDA-MB-231 (Fig. 5, e-g) and BT-549 cells (Fig. 5, l-n). Especially, 5-HT7 and FOXM1 expressions were significantly suppressed in the cells transfected with 100 nM 5-HT7-siRNA. These results indicated that 5-HT7 is involved in expression of FOXM1 in TNBC cells.
3. 6. 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 [34-36]. 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 . 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 significantly 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 . We found that Cyclin-D1 expression was significantly 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.
3. 7. 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 findings indicate that 5-HT7-induced TNBC cell proliferation is mediated by FOXM1expression.