Tumor Suppressor ENDOD1 Slows Colorectal Cancer Growth in Cell and Mouse Studies

A gene called ENDOD1, which is consistently underactive in colorectal cancer tissue, has emerged as a candidate tumor suppressor in a new study published in the journal Cell Division. Researchers report that when they forced colorectal cancer cells to produce extra ENDOD1, the cells became less viable, less able to migrate and invade, and more prone to apoptosis, the programmed cell death process that cancer cells often evade. Conversely, silencing the gene in the same cells produced the opposite effects, boosting proliferation, motility, and invasiveness. In mice carrying transplanted tumors, ENDOD1 overexpression slowed tumor growth, reduced the proliferation marker Ki67, and increased tumor cell death. The work is entirely preclinical, based on laboratory cell lines and a mouse xenograft model, so the findings describe mechanisms in experimental systems rather than demonstrated benefits for patients.

The team, led by Yichi Zhang and Dehua Hu, began with a computational screen rather than a hypothesis about ENDOD1 specifically. They analyzed four publicly available gene expression datasets from the Gene Expression Omnibus: GSE41657, GSE8671, GSE41328, and GSE75548, each comparing colorectal cancer tissue with normal tissue. Using a threshold of an absolute log2 fold change greater than 0.8 and an adjusted p-value below 0.05, they identified hundreds to more than a thousand differentially expressed genes in each dataset, including 1,348 decreased and 982 increased genes in GSE41657 alone. Cross-referencing the four datasets narrowed the field to 31 genes commonly decreased and 55 commonly increased across all of them.

To prioritize which of these shared genes might matter for patient outcomes, the researchers turned to the TCGA-COAD database and examined correlations between gene expression and overall survival. Four downregulated genes, CITED2, NR3C2, FAM46C, and ENDOD1, were significantly associated with protective effects on survival, each with a hazard ratio below 0.90 and a p-value below 0.05. ENDOD1 showed the smallest hazard ratio of the group, 0.82, with a 95 percent confidence interval of 0.57 to 1.2. Notably, that confidence interval crosses 1, meaning the survival association, while statistically significant by the study’s threshold, carries considerable uncertainty. Five upregulated genes, including TIMP1 and S100A11, were flagged as risk factors. ENDOD1 was confirmed as downregulated in all four GEO datasets.

The bioinformatic signals were then tested experimentally. Using RT-qPCR and Western blot, the researchers measured ENDOD1 messenger RNA and protein in four colorectal cancer cell lines, HCT116, HT-29, SW620, and SW480, alongside the normal colonic epithelial cell line NCM460. ENDOD1 levels were dramatically lower in all four cancer cell lines at both the RNA and protein levels, with the lowest expression observed in HT29 cells. That cell line was therefore selected as the workhorse for the functional experiments that followed, a choice that concentrates the study’s mechanistic claims on a single genetic background.

The functional tests combined loss- and gain-of-function approaches. The team transfected HT29 cells with an ENDOD1 overexpression plasmid or, separately, with two small interfering RNAs designed to knock the gene down. A CCK-8 assay tracking viability over 0, 24, 48, and 72 hours showed that ENDOD1 overexpression significantly reduced cell viability, while knockdown significantly increased it. Flow cytometry using Annexin V-FITC/PI staining revealed that overexpression markedly enhanced apoptosis, both with and without exposure to the chemotherapy drug 5-fluorouracil, whereas knockdown reduced 5-FU-induced apoptosis. One nuance deserves mention: knocking down ENDOD1 did not significantly change the basal apoptotic rate under non-stress conditions, which the authors attribute to the low baseline level of apoptosis in unstressed cells.

Migration and invasion assays told a consistent story. A scratch test, in which a sterile pipette tip wounds a confluent cell monolayer and closure is photographed over 24 hours, showed that ENDOD1 overexpression diminished migratory capacity while knockdown increased it. Transwell chambers with 8-micrometer pores coated in Matrigel produced parallel results for invasion, with overexpression reducing the number of cells that penetrated the matrix and knockdown increasing it. Together, these in vitro results support the characterization of ENDOD1 as a suppressor of the malignant behaviors that make colorectal cancer dangerous, particularly recurrence and metastasis.

To test whether the effect extended beyond the dish, the researchers used a xenograft model in which HT29 cells stably infected with ENDOD1-overexpressing lentivirus or a blank control lentivirus were injected subcutaneously into twelve five-week-old male BALB/c nude mice, six per group. After four weeks, tumors in the ENDOD1 group were dramatically smaller in volume and weight than controls. Immunohistochemistry showed reduced Ki67 staining, a marker of actively dividing cells, and TUNEL staining revealed a notably higher rate of apoptosis in the ENDOD1-overexpressing tumors. These animal results align with the cell culture data, though xenograft models implanted with a single human cell line into immunodeficient mice capture only a narrow slice of tumor biology and cannot recapitulate the immune interactions or genetic diversity of human colorectal cancer.

The mechanistic core of the study concerns the Wnt/beta-catenin pathway, a signaling cascade whose hyperactivation is a well-established driver of colorectal cancer development and a long-sought therapeutic target. Guided by gene set enrichment analysis of genes correlated with ENDOD1 expression in TCGA-COAD, the team examined key pathway components by Western blot. Silencing ENDOD1 significantly increased levels of beta-catenin, c-Myc, and cyclin D3, while overexpression repressed all three proteins. Because beta-catenin is the pathway’s central signal transducer and c-Myc and cyclin D3 are downstream proliferation drivers, the pattern indicates that ENDOD1 restrains Wnt/beta-catenin signaling activation in these cells.

The most direct evidence for causality came from a rescue experiment. The researchers treated ENDOD1-overexpressing HT29 cells with Laduviglusib, a potent GSK-3 alpha/beta inhibitor at 10 micromolar that pharmacologically activates Wnt/beta-catenin signaling. The drug partially restored the expression of beta-catenin, c-Myc, and cyclin D3 that ENDOD1 overexpression had suppressed, and it partially reversed the overexpression-induced reductions in cell viability, migration, and invasion, as well as the increase in apoptosis. The word partially is important: reactivating the pathway mitigated but did not abolish ENDOD1’s suppressive effects, suggesting that ENDOD1 acts through Wnt/beta-catenin inhibition at least in part, with other mechanisms likely contributing.

The authors also connect their findings to ENDOD1’s known biology. The gene encodes endonuclease domain containing 1, a member of the nuclease family of enzymes that hydrolyze phosphodiester bonds in nucleic acids and contribute to mutation avoidance, DNA repair, and programmed cell death. Prior studies have linked reduced ENDOD1 to increased local invasiveness in soft tissue tumors and decreased expression in high-grade, lymph node-metastatic prostate cancer, and ENDOD1 has been reported as an independent prognostic factor in prostate and pancreatic ductal cancers. The authors speculate that ENDOD1’s tumor-suppressive role in colorectal cancer may relate to preserving genomic integrity, but they present this as a plausible hypothesis rather than a demonstrated mechanism. Taken together, the study positions ENDOD1 as a potential biomarker and research target for colorectal cancer, while the path from cell lines and xenografts to any clinical application in diagnosis or therapy remains long and unproven.

Subject of Research: ENDOD1 as a tumor suppressor in colorectal cancer and its inhibition of Wnt/beta-catenin signaling

Article Title: Overexpression of ENDOD1 inhibits proliferation, migration, and invasion of colorectal cancer

Article References: Zhang, Y., Li, N., Zhang, H., & Hu, D. (2026). Overexpression of ENDOD1 inhibits proliferation, migration, and invasion of colorectal cancer. Cell Division, 21(1), Article 16. https://doi.org/10.1186/s13008-026-00181-5

Image Credits: AI Generated

DOI: 10.1186/s13008-026-00181-5

Keywords: ENDOD1, colorectal cancer, Wnt/beta-catenin signaling, tumor suppressor, HT29 cells, xenograft mouse model, bioinformatics, GEO datasets, apoptosis, gene expression, RT-qPCR, Western blot