EpCAM deficiency causes the premature ageing of intestinal stem cells via EGFR/SP1/mTORC1 pathway

Dear Editor, We reported a precise molecular mechanism underlying the longevity of intestinal stem cells (ISCs) maintained by EpCAM via the EGFR/SP1/mTORC1 pathway. EpCAM is highly expressed in ISCs,1 and most patients of congenital tufting enteropathy (CTE), caused by EpCAM mutations,2, 3 die of intestinal failure.4 EpCAM deficiency induces premature ageing of ISCs via downregulating TSC1 to hyperactivate the mTORC1 pathway.5 However, upstream signals through which EpCAM regulates the expression of TSC1 remain unknown. As previous report,5 EpCAM−/− mice showed no significant differences in appearance compared to wild type (WT) littermates at the E18.5 stage (Figure S1A,B). However, the mutant pups started to have diarrhoea after birth and most died within 1 week. The tufting and erosion of the intestinal villi were observed in mutants at both E18.5 and P3 stages (Figures S1C,D and S2A,B). Tert, encoding the protein component of telomerase, was significantly downregulated but γH2AX, the DNA damage accumulation-associated protein, was considerably increased in the E18.5 mutant small intestines (Figure S1E−G), confirming the premature ageing of these tissues. SP1 is involved in various tissue and cellular senescence processes.6 At the E18.5 stage, the expression and activity of SP1 were reduced in the mutant small intestines (Figure 1A,B). The p-SP1 was predominantly present in the nucleus of epithelial cells in the inter villi region of the duodenum and decreased in the mutants at both E18.5 and P3 stages (Figures S3A and S4A−F). The SP1 inhibitor Mithramycin A (MitA) reduced the expression and activity of SP1 in Caco-2 cells (Figures 1C,D and S3B,C). With the inhibition of SP1, TSC1 and TSC2 downregulated, and the activation of the mTORC1 pathway dramatically increased (Figures 1E and S3D). The TERT significantly decreased, but γH2AX increased with the MitA administration (Figure 1F−H). Subsequently, Caco-2 cells were simultaneously treated with MitA and rapamycin (RAP). The p-SP1 and SP1 were significantly decreased in MitA and MitA+RAP groups (Figures 1I,K and S3E). In the MitA+RAP group, the activation of the mTORC1 pathway was markedly lower than in the MitA group (Figures 1J and S3E). The TERT was increased but γH2AX was decreased in the MitA+RAP group compared with the MitA group (Figures 1K,L and S3E). The naringenin (NAR), an effective activator of SP1,7 was administrated to the pregnant females to activate SP1 in the intestines of EpCAM−/− embryos (Figure S5A). The duodenum and jejunum showed improved intestinal villi tufting and breakage in the NAR-treated mutants (Figure S5B). The expression and activity of SP1 were significantly restored in the small intestines of the EpCAM−/−+NAR group (Figures 2A,B and S5C). After administration of NAR, TSC1 was significantly upregulated but the activation of the mTORC1 pathway noticeably decreased in the mutant intestines (Figures 2C,D and S5D). With NAR treatment, there was a certain tendency for a rebound of the transcription of Tert in the mutant intestines (p = 0 .07) (Figure 2E). Again, TERT was significantly increased but γH2AX was decreased in the small intestines of NAR-treated EpCAM−/− mice compared with control mutants (Figures 2F and S5C). Sirtuins decrease in various ageing cells and tissues.8 The NAR significantly rescued SIRT1, SIRT3, SIRT6 and SIRT7 in the mutant intestines (Figure 2G,H). The transcriptions of Sirt3, Sirt4, Sirt5 and Sirt7 were significantly restored in the mutant small intestines with the NAR administration (Figure S5E). In tumour cells of epithelial origin, interactions between EGFR and EpCAM have been demonstrated.9 The expression and activity of EGFR were increased in the mutant small intestines at the E18.5 stage (Figure 3A,B). Therefore, gefitinib was selected to reduce the hyperactivation of EGFR in the intestines of EpCAM−/− mice. Gefitinib significantly decreased the ratio of p-EGFR/EGFR in the small intestines of EpCAM−/− mice (Figures 3C,D and S6A). The TERT was increased in the gefitinib-administered EpCAM−/− mice, and the γH2AX was reduced accordingly (Figure 3E−G). EGFR can activate the mTORC1 pathway.10 The expression and activity of SP1 were significantly rebounded in the small intestines of gefitinib-treated EpCAM−/− mice (Figures 4A,B and S6B). The expression of TSC1 was significantly restored after the administration of gefitinib, but the activation of the mTORC1 pathway decreased in the small intestines of treated mutants (Figures 4C,D and S6C). A significant rebound of SIRT1 and SIRT5 and an increasing trend of SIRT6 and SIRT7 were confirmed in the small intestines of EpCAM−/− mice after administration of gefitinib (Figures 4E and S6D). The transcriptions of Sirt1, Sirt3, Sirt4, Sirt5 and Sirt7 were restored in the small intestines of gefitinib-administrated EpCAM−/− mice (Figure S6E). In conclusion, loss of EpCAM causes the premature ageing of ISCs via EGFR/SP1/mTORC1 pathway (Figure 4F). EpCAM deficiency causes the hyperactivation of EGFR in the ISCs. Then, the sustained hyperactivation of EGFR decreases the activity of SP1 which controls the expression of Tsc1. Subsequently, the downregulated TSC1 induces the hyperactivation of mTORC1 and finally causes the premature ageing of the ISCs. Our findings put forward a new viewpoint on the interactions between EpCAM and EGFR in the membranes of stem cells and even tumour cells and provide potential targets for the CTE therapy. K.L.: Methodology, investigation, writing—original draft preparation. C.X.: Investigation, funding acquisition. L.L.: Methodology, validation. Y.W.: Methodology, visualization. J.C.: Software. C.L.: Visualization. Z.P.: Methodology. X.L.: Methodology. G.C.: Writing—review and editing, funding acquisition. Z.L.: Supervision, conceptualization, funding acquisition. Y.Y.: Supervision, investigation, funding acquisition. The authors have nothing to report. The authors report no conflicts of interest. This work was supported by the National Natural Science Foundation of China (82171855); the Key Field Special Project for Colleges and Universities of Guangdong Province (Biomedical and Health) (2023ZDZX2030); the Natural Science Foundation of Shenzhen (JCYJ20210324120212033); the Guangdong Basic and Applied Basic Research Foundation (2021A1515012383); the Scientific Research and Technological Development Plan of Nanning City (20213024); the Science and Technology Project of Qing-Xiu District of Nanning City (2020025); the Science and Technology Project of Jiang-Nan District of Nanning City (20220620-8, 20230715-07); and the Key Research and Development Plan of Scientific Research and Technology in Liang-Qing District of Nanning City (202213, 202216, 202311). All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Guangdong Pharmaceutical University. Datasets are available from corresponding authors on reasonable request. 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