Serotonin acts through YAP to promote cell proliferation: mechanism and implication in colorectal cancer progression

Abstract

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a key messenger that mediates several central and peripheral functions in the human body. Emerging evidence indicates that serotonin is critical in tumorigenesis, but its role in colorectal cancer remains elusive. Herein, we report that serotonin transporter (SERT) transports serotonin into colorectal cancer cells, enhancing Yes-associated protein (YAP) expression and promoting in vitro and in vivo colon cancer cell growth. Once within the cells, transglutaminase 2 (TG2) mediates RhoA serotonylated and activates RhoA-ROCK1/2 signalling to upregulate YAP expression in SW480 and SW1116 cells. Blocking SERT with citalopram reversed the serotonin-induced YAP expression and cell proliferation, inhibiting serotonin's effects on tumour formation in mice. Moreover, SERT expression was correlated with YAP in pathological human colorectal cancer samples and the levels of 5-HT were highly significant in the serum of patients with colorectal cancer. Together, our findings suggested that serotonin enters cells via SERT to activate RhoA/ROCK/YAP signalling to promote colon cancer carcinogenesis. Consequently, targeting serotonin-SERT-YAP axis may be a potential therapeutic strategy for colorectal cancer. Video abstract.

Keywords: Colorectal cancer; Serotonin; Serotonin transporter; Serotonylation; YAP.

Fig. 1

5-HT proliferates colon cancer cells through enhancing YAP expression. A 5-HT incubated SW480 and SW1116 cells for 48 h at their indicated concentrations. Furthermore, the CCK-8 assay was used to evaluate the proliferation of cancer cells. *, P < 0.05; **, P < 0.01. B EdU staining (100 ×) determined the cell proliferation of SW480 and SW1116 cells after 10 μM 5-HT stimulation was determined. C 5-HT was used to treat the cells at 0.1 μM-100 μM concentrations for 4 h or with 10 μM 5-HT from 30 to 240 min; a western blot test was performed to determine changes in YAP’s expression of its target proteins in SW480 and SW1116 cells. D Cells were pre-treated with 1 μM verteporfin 2 h before 10 μM 5-HT stimulation, and 5-HT was combined with a CCK-8 assay to induce proliferation abilities of SW480 and SW1116 cells. *, P < 0.05; **, P < 0.01. E After RNAs were transferred and YAP expression was knocked down in SW480 and SW1116 cells, 5-HT induced cell proliferation was used in combination with CCK-8 assay for this experiment. *, P < 0.05; **, P < 0.01

Fig. 2

SERT transports extracellular 5HT entry into colon cancer cells. A CCK-8 assay treated the cells with the broad spectrum 5-HTR inhibitor asenapine (100 μM) and SERT antagonists citalopram (100 μM) or fluvoxamine (100 μM) for 2 h before 5-HT stimulation. In succession to this procedure, cell proliferation was detected by the CCK-8 assay. *, P < 0.05; **, P < 0.01. B Moreover, a western blot analysis of YAP and Cyr61 expression in SW480 and SW1116 cells were treated with 100 μM asenapine or SSRIs citalopram, and fluvoxamine for 2 h before using 5-HT stimulation. C A western blot test was used to stimulate YAP and Cyr61 after knocking down SERT expression in SW480 and SW1116 cells

Fig. 3

TG2 mediates intracellular 5-HT serotonylates and activates RhoA to promote YAP in colon cancer cells. A After siRNA transfection downregulated TG2 expression in SW480 and SW1116 cells, a western blot was used to analyse YAP and Cyr61 expression in cells treated before with 10 μM 5-HT. B After downregulating RhoA expression in SW480 and SW1116 cells, YAP and Cyr61 expressions in cells were treated with 10 μM 5-HT. For this procedure, a western blot test modelled these expressions. C GST pull-down analysis was used to detect the levels of total and active RhoA in cells stimulated with 5-HT (10 μM) or pre-treated with 100 μM citalopram before 5-HT stimulation. D Immunoprecipitation was performed to analyse the expression of RhoA, 5-HT in cells stimulated with 5-HT (10 μM) or pre-treated with 100 μM citalopram before 5-HT stimulation

Fig. 4

ROCK1/2 but not Lats1/2 mediates 5-HT-induced YAP expression in colon cancer cells. A A western blot test was used to analyse YAP and Cyr61 expression in cells treated with 10 μM 5-HT. This procedure was done after Lats1 expression was knocked down in SW480 and SW1116 cells. B After Lats2 expression was knocked down in SW480 and SW1116 cells, YAP and Cyr61 expressions in cells treated with 10 μM 5-HT were analysed by western blot. C Cells were treated with ROCK1/2 antagonists Y27632 or SLx-2119 (10 μM) for 2 h before 10 μM 5-HT stimulation; a western blot was performed to detect the expression of YAP and Cyr61 in SW480 and SW1116 cells

Fig. 5

5-HT promotes primary colon carcinogenesis in mice. A AOM combined with DSS in indicative groups was used to treat tumour images in the mice’s intestinal tissues. B Tumour numbers in the indicated groups of mice. *, P < 0.05; **, P < 0.01. C Tumour volume in the indicated groups of mice. *, P < 0.05; **, P < 0.01. D Immunohistochemistry was performed to analyse YAP expression in colorectal carcinoma tissues of the indicated group of mice. *, P < 0.05; **, P < 0.01. E ELISA analysis detected the serum of the mice’s indicated group’s contents of 5-HT. *, P < 0.05; **, P < 0.01

Fig. 6

YAP and SERT expression levels are elevated in human colorectal cancer. A Immunohistochemistry was used to detect YAP and SERT expression in a representative CRC sample and a matched adjacent normal intestinal tissue (original magnification, 200 ×). B ELISA analysis detected the 5-HT content in the serum of patients suffering from CRC before and after surgery. **, P < 0.01. C ELISA analysis detected 5-HT content in the patients’ serum with multiple metastatic CRC and that of normal people. **, P < 0.01

Fig. 7

Schematic representation of the proposed mechanism by which 5-HT modulates signalling pathways in colon cancer. A In colon cancer cells, extracellular 5-HT is transported into cells by SERT located in the cytomembrane and then transamidates and activates RhoA via serotonylation regulated by TG2. Subsequently, ROCK1/2 is activated, leading to the expression of YAP and its target proteins. B When SERT function is blocked by specific antagonists (such as citalopram), 5-HT intracellular transportation is weakened to some degree, blocking the cellular signalling transduction and colon cancer cell proliferation induced by 5-HT. This strategy suggests that targeting 5-HT signalling may be feasible for treating colon cancer