Targeting SERT Promotes Tryptophan Metabolism:Mechanisms And Implications In Colon Cancer Treatment

Objective:5-HT(5-hydroxytryptamine,serotonin)signal transduction is closely related to tumorigenesis and tumor progression.Targeting serotonin transporter(SERT)to block serotonin cellular uptake is reported to be antineoplastic in various tumors.However,little is known about the alternation and the homeostasis of 5-HT metabolism in the absence of SERT,and this will undoubtedly affect the anti-tumor efficacy of targeting SERT.The present study will take colon cancer which is closely related to this field as an example to explore the changes in 5-HT metabolism after targeting SERT to inhibit the exogenous 5-HT pathway of colon cancer cells and provides novel mechanistic insights into the regulation of serotonin homeostasis in colon cancer as well as suggests innovative strategies for SSRIs-based treatment of colon cancer.Materials and Methods:1.The m RNA level of tryptophan transporters(SLC1A5,SLC7A5),key metabolic enzymes in Kyn pathway(TDO2,AFMID),the synthetase in 5-HT pathway(TPHI)was detected after interfering SERT expression by si RNA and inhibiting SERT activity with sertraline in SW480 and HCT116 colon cancer cell lines.Western blot was used to detect the protein expression levels of transporters SLC1A5 and SLC7A5 after si SERT and sertraline treatment.The intracellular concentration of the three key metabolites Trp,5-HT and Kyn in Trp catabolic pathway was determined by LCMS/MS.2.AOM/DSS was utilized to establish a primary colon cancer model in SERTKO mice and SERT-WT mice.By calculating the tumor volume and number,the effect of SERT knockout on colon cancer was observed.RT-PCR detection was performed in colon cancer tissues of AOM/DSS group mice(SERT-WT and SERT-KO)and normal colon tissues of control group(SERT-WT and SERT-KO)to identified the m RNA level of transporters(SLC1A5,SLC7A5)and key enzymes in tryptophan metabolism pathway(AFMID,TPH1).The concentration of metabolites(Trp,5-HT and Kyn)in mouse plasma was observed by LCMS/MS.AOM/DSS was used to induce a primary colon cancer model in SERT-WT mice,and then divides into two groups which were treated with sertraline(30mg/kg)and DMSO respectively.Tumor volume and number were calculated,and the effect of SERT inhibitor sertraline on primary colon cancer in mice was observed.The m RNA level of SLC1A5,SLC7A5,AFMID,TPH1 is detected by q PCR and the concentration of Trp,5-HT,and Kyn is detected by LCMS/MS.3.SW480 and HCT116 cells were starved in serum-free medium for 24 h,and then stimulated with 5-HT.The effect of 5-HT on the protein level of p-mTOR,p-S6 K and SLCA5 was performed by Western Blot.After SERT interference or sertraline treatment(time and concentration gradient)in colon cancer cells,western blot was utilized to detect the changes in the protein level of p-mTOR,p-S6 K and SLC1A5 in the mTOR pathway.Colon cancer cells were treated with the mTOR activator MHY1485 and the inhibitor rapamycin,and the expression of SLC1A5 was identified by Western blot.5-HT was re-supplemented after interference and suppression of SERT,and protein level of p-S6 K and SLC1A5 was detected by Western blot.4.Co-immunoprecipitation(IP)and Western blot were used to detect the presence of serotonylated mTOR and the changes in serotonylated mTOR in colon cancer cells after 5-HT treatment.SW480 and HCT116 cells were pretreated with MDC(TG2inhibitor)or sertraline and then stimulated with 5-HT.IP and Western blot were used to detect the changes of serotonylated mTOR.Cells were transfected with TG2 si RNA or MDC and then stimulated with 5-HT.The protein expression level of p-mTOR,pS6 K and SLC1A5 was estimated.Western blot was utilized to detect the changes of mTOR/S6 K pathway after treatment with 5-HT receptor broad-spectrum inhibitor(Anasepin).5.After starving cells with serum-free and trp-free medium and followed by adding 5-HT and tryptophan exogenously,immunofluorescence(IF)test was used to detect the intracellular localization of 5-HT.IF test detected the intracellular localization of 5-HT after SERT inhibition.IF test detected the co-localization of 5-HT and lysosome after exogenous addition of 5-HT,and the changes of co-localization after sertraline and MDC treatment for 30 min.6.AOM combined with DSS was used to induce primary colon cancer in C57BL/6 mice.The mice’s diet was changed from a regular diet to control(2mg)or double tryptophan(DT)diet(4mg).By calculating the tumor volume and number,the effect of increased tryptophan metabolism on colon cancer was observed.The subcutaneous allograft tumor model was established with the murine colon cancer cell line CT-26.One week before planting the tumor,the mouse diet was changed to a control or DT/TTR(complete tryptophan restriction)diet/PTR(partial tryptophan restriction)diet.At last,the tumor was dissected.The tumor volume and weight were calculated to observe the effect of increasing and limiting tryptophan metabolism on subcutaneous transplanted tumors in mice.7.The protein expression of SLC1A5,SLC7A5,IDO1,AFMID,TPH1,SERT,MAOA,MAOB,and TG2 of colon cancer tissues and the corresponding adjacent tissues in the tissue array was observed by immunohistochemical staining(IHC).8.The effect of SERT inhibition on the proliferation of colon cancer cells SW480 and HCT116 in the presence or absence of Trp or trametinib was measured by the CCK8 experiment.The colony formation assay was used to observe the synergetic effect of sertraline and trametinib.HCT116 cells were utilized to construct a nude mouse xenograft tumor model to observe the combined therapeutic effect of sertraline and diet-restricted tryptophan/trametinib in vivo.AOM/DSS-induced colon cancer mouse model was established in SERT-KO and SERT-WT mice to determine the synergistic effect of SERT knockout with dietary Trp restriction.Results:1.Inhibition of SERT promotes the absorption and catabolism of tryptophan in colon cancer cells to keep the cellular content of serotonin.2.Inhibition of SERT activates Trp catabolism in primary colon cancer mouse model.3.5-HT activates the mTORC1/S6 K signaling pathway in colon cancer cells.Inhibition of SERT reduces the concentration of cellular serotonin through inhibiting the reuptake of serotonin,thereby inhibiting mTOR signaling to promote Trp uptake and catabolism.This process can be partly reversed by 5-HT supplementation.4.There is serotonylation of mTOR in colon cancer cells,and its level can be increased after 5-HT treatment.This process can be reversed by TG2 inhibitor(MDC)or sertraline treatment.The activation of the mTOR pathway by 5-HT is weakened after TG2 interference or MDC treatment rather than 5-HT receptor broad-spectrum inhibitor.5.Endogenous and exogenous 5-HT has distinct subcellular locations.The endogenous 5-HT synthesized by Trp is mainly located around the nucleus,while the exogenous 5-HT transported into the cell through SERT is extensively distributed in the periphery of the cytoplasm.The inhibition of SERT by si RNA or sertraline treatment can result in the increase of 5-HT localization around the nucleus.Exogenous 5-HT has better co-localization with lysosomes,and the co-localization is significantly reduced after a short period of sertraline and MDC treatment,which implying that mTORC1 may play a role in sensoring the change of cellular serotonin concentration and regulate the balance of endogenous and exogenous 5-HT pathway.6.Enhancing tryptophan metabolism can promote tumor growth in AOM/DSS primary colon cancer mouse model and in subcutaneous xenograft model,while weakening tryptophan metabolism can inhibit tumor growth in subcutaneous xenograft models.7.The immunohistochemical analysis on the micro array of colon cancer(66 pairs of normal and tumor tissues)revealed that SLC1A5,SLC7A5,IDO1,AFMID TPH1,SERT,MAOA,MAOB and TG2 are all over expressed in cancer tissues.8.The combination of sertraline and restricted tryptophan/trametinib shows synergistic anti-tumor activity in vitro and in vivo.Conclusions:1.Inhibition of SERT promotes Trp uptake and catabolism both in vivo and in vitro.2.5-HT activates mTOR through serotonylated modification.Targeting SERT reduces the intracellular 5-HT level by inhibiting the reabsorption of 5-HT,which results in the weakening of mTOR activity,and mTOR increases the uptake and catabolism of Trp to maintain intracellular 5-HT levels and its own activity.3.Endogenous 5-HT and exogenous 5-HT have different subcellular localization.Exogenous 5-HT has better co-localization with lysosomes.Inhibition of SERT may reduce serotonylated modification of mTOR by inhibiting the co-localization of 5-HT and lysosomes.4.Enhanced Trp metabolism can promote the development of colon cancer and limited Trp metabolism can inhibit the development of colon cancer.5.The combination of targeted SERT and tryptophan restriction /trametinib synergistically inhibits colon cancer cell proliferation in vitro and tumor growth in vivo.