Robust regulation of endogenous TCF7L2 expression in transgenic mice.

(a) Schematic representation of the Tet-inducible system to regulate endogenous Tcf7l2 expression in double transgenic mouse. The reverse tetracycline trans-silencer (rTetR-KRAB) is constitutively expressed from the ROSA26 genomic region. Treatment with doxycycline (Dox) causes rTetR-KRAB to bind tetracycline response element (TRE) and transcriptionally repress Tcf7l2. Tcf7l2 expression is restored upon cessation of doxycycline treatment. (b-h) WT (Rosa26rTetR-KRAB/WTTcf7l2WT/WT) and KI (Rosa26rTetR-KRAB/WTTcf7l2TRE/TRE) mice were treated with either high or low dose doxycycline (dox) for various time durations. (b) Experimental timeline for high dose doxycycline treatment and withdrawal. Tcf7l2 (c) mRNA and (d) protein expression in the small intestine following doxycycline treatment and withdrawal as outlined in (b). Data are mean ± SEM. Statistical significance was assessed by Student’s t-test. All experimental groups n = 3. ***P < 0.001 and ns, not significant. Scale bar 50μm. (e-f) Mice were continuously treated with high dose doxycycline for ten days. (e) Body weight with respect to day 0 and (f) H&E staining of small intestine on day 10. Data are mean ± SEM. WT n = 3 and KI n = 6. Scale bar 50μm. (g-h) To partially repress Tcf7l2, mice were continuously treated with low dose. (g) Relative Tcf7l2 mRNA expression in the indicted tissues on day 5 and (h) H&E staining of small intestine on week 6. Data are mean ± SEM. Statistical significance was assessed by Student’s t-test. All experimental groups n = 3. **P < 0.01 and ns, not significant. Scale bar 50μm. eAT, epididymal adipose tissue. iAT, inguinal adipose tissue. SI, small intestine.

Tcf7l2 repression rescues colon cancer mice from fatal cachexia.

(a) Genotypes of control and experimental mice at the three loci indicated. (b) Experimental timeline for colon tumor development and doxycycline treatment. (c) Body weight with respect to day -15. (d) Mass of the indicated tissues from mice undergoing two days (Col-WT) or five days (noCol-WT, noCol-KI and Col-KI) of doxycycline treatment. (e) Survival analysis of mice given continuous doxycycline treatment (continuous dox) or when doxycycline treatment ceased on day 14 (stop dox). Data are mean ± SEM. Statistical significance was assessed one-way ANOVA with Bonferroni test. noCol-WT n = 5, noCol-KI n = 6, Col-WT n = 4, Col-KI n = 4. All experimental groups n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 and ns, not significant. DSS, dextran sodium sulphate.

Colon tumors continue to progress despite recovery in mice.

Mice were treated with doxycycline for various lengths of time, as indicated. (a) Relative mRNA expression of Tcf7l2 and Wnt target genes in tumors and the adjacent normal tissue. (b) Representative images of longitudinally opened colon. Scale bar 1cm. (c) Quantification of tumor burden and (d) distribution of tumors by size. (e) H&E staining of tumors. Arrows indicate adenocarcinoma growth that has invaded the submucosa. Scale bar 100μm. (f- g) Gene Set Enrichment Analysis (GSEA) based on bulk RNA-Seq of tumors. Doxycycline treatment was initiated several days early so that mice were not yet moribund at the point of euthanasia. Shown are Hallmark gene sets with false discovery rate < 0.25. Bars are colored according to biological function of the gene set, as indicated. NES, normalized enrichment score. Data are mean ± SEM. Statistical significance was assessed by one- way ANOVA with Bonferroni test. APC-WT 2 days (a) n = 3 and (c) n = 4, APC-KI 5 days n = 4, APC-KI 6 weeks n = 5. **P < 0.01; ***P < 0.001 and ns, not significant.

TCF7L2 regulates the expression of numerous atrophy genes in the gastrocnemius muscle.

(a-d) Relative mRNA expression of (a) Tcf7l2, (b) ubiquitin-proteasome system genes, (c) autophagy-lysosome system genes and (d) myostatin in the gastrocnemius. Data are mean ± SEM. Statistical significance was assessed one-way ANOVA with Bonferroni test. noCol-WT n = 5, noCol-KI n = 6, Col-WT n = 4, Col-KI n = 4. **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.

TCF7L2 is essential for early tumourigenesis in the small intestine.

(a) Experimental timeline for small intestine tumor development and doxycycline treatment. (b) Body weight with respect to experimental day 0. (c) Representative photos of longitudinally opened small intestine. Arrows indicate tumor lesions. Scale bar 5 mm. (d) Quantification of tumor burden. (e) H&E staining of small intestine. Scale bar 100μm. Data are mean ± SEM. Statistical significance was assessed one-way ANOVA with Bonferroni test. noSI- WT n = 8, noSI-KI n = 4, SI-WT n = 4, SI-KI n = 3. ***P < 0.001; ****P < 0.0001 and ns, not significant.

Tcf7l2 repression rescues mice from fatal cachexia without affecting small intestine tumor growth.

(a) Experimental timeline for tumor development and doxycycline treatment. (b) Body weight with respect to day-10. (c) Relative mRNA expression of Tcf7l2 in tumors and the adjacent normal tissue. (d) Representative photos of longitudinally opened small intestine. Arrows indicate tumor lesions. Scale bar 5 mm. (e) Quantification of tumor burden. (f) H&E staining of small intestine. Scale bar 100μm. (g) Mass of the gastrocnemius and adipose tissues. (h-i) Relative mRNA expression of (h) Tcf7l2 and (i) muscle atrophy genes in the gastrocnemius. Data are mean ± SEM. Statistical significance was assessed one-way ANOVA with Bonferroni test. noSI-WT n = 5, noSI-KI n = 5, SI-WT n = 5, SI-KI n = 4. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.