Fig 2: DACH1 suppression inhibits colon cancer cell growth in vitro and in vivo. The efficiency of DACH1 knockdown in colon cancer cell lines was confirmed by western blotting (a). Cells with knockdown of DACH1 displayed increased inhibition of cell growth compared with control cells (b and c, experiments were performed in triplicate). Representative images of xenografts at the end of the study (d) showing decreased growth in HCT116-shDACH1 cells compared with the control, including tumour volumes (e) and tumour weights (f). Knockdown of DACH1 significantly reduced the number of spheres formed by HCT116 cells (g and h, scale bar=200 µm). IHC assays showed weaker CD166, OLFM4 and SOX2 staining in xenografts in which DACH1 was knocked down than in the controls (i). For DACH1 staining images, the scale bars=50 µm, and for CD166, OLFM4 and SOX2 staining images, the scale bars=20 µm. For 2c, 2e, 2f and 2 g, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. Error bars: mean±SD.

Fig 3: DACH1 marks crypt base cells in intestines and predicts poor outcomes in colorectal cancer patients. DACH1 expression in the mouse small intestine (a) and large intestine (b); scale bars=20 µm. In the mouse small intestine, DACH1 is expressed in crypt base cells interspersed between Paneth cells. Lysozymes (a marker of Paneth cells) were stained red, and DACH1 was stained green, which merged into cyan with DAPI (blue); scale bar=20 µm (c). DACH1 is also expressed in the human large intestine; scale bar=20 µm (d). DACH1 mRNA is overexpressed in colorectal cancer tissues compared to adjacent normal tissues, as detected by qRT-PCR (e), and IHC revealed that the expression of DACH1 increased in all stages of CRC when compared with the normal tissue (f) (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test; error bars: mean±SD). Elevated DACH1 expression at the invasive front of the tumour lesion; scale bar=100 µm (g). Kaplan-Meier survival analyses based on DACH1 expression for overall survival (h, P < 0.001, log-rank test, between high level and low level of DACH1) and disease-free survival (i, P < 0.001, log-rank test, between high level and low level of DACH1).

Fig 4: DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFß3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 µm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse immunoprecipitation was confirmed with an anti-SMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 µm. For 5d, 5e, 5k, 5 m and 5n, **P < 0.01, *P < 0.05, Student's t-test. Error bars: mean±SD.

Fig 5: DACH1 overexpression enhances the growth of organoids derived from normal colon crypts and colorectal adenomas. DACH1 overexpression efficiency was verified by qRT-PCR (experiments were performed in triplicate) (a) and western blot (b). c. Overexpression of DACH1 enhanced the sphere formation of organoids derived from normal colon crypts (colonoid) compared with the control; scale bars=200 µm d. Overexpression of DACH1 significantly enhanced colonoid proliferation compared with the vector group. e. Overexpression of DACH1 significantly enhanced the colonoid formation rate compared with the control. DACH1 overexpression was verified by qRT-PCR (experiments were performed in triplicate) and western blot (f and g). h. Adenoma organoids with DACH1 overexpression expanded to almost twice the size of the control organoids; scale bars=200 µm. i. Overexpression of DACH1 significantly enhanced the proliferation of organoids derived from colorectal adenoma compared with the control. j. The adenoma organoid formation efficiency was improved by DACH1 overexpression. k-l. The organoid areas of DACH1-overexpressing organoids were within the lower quartile more than the control; scale bars=200 µm. m-n. The average adenoma organoid areas in DACH1-overexpressing organoids were significantly larger than those in the control; scale bars=100 µm. For 4a, 4d, 4e, 4f, 4i, 4j and 4n, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. Error bars: mean±SD.