A RAC-GEF network critical for early intestinal tumourigenesis

Abstract

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2^-/- Vav3^-/- Tiam1^-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

Fig. 1. Loss of Rac1 perturbs villus homeostasis.

a Images showing H&E, BrdU incorporation and Cleaved caspase-3 IHC in Vil-CreER^T2 (wild-type), Vil-CreER^T2 Rac1^fl/fl (Rac1^fl/fl) mice day 3 post induction and Vil-CreER^T2 Rac1^fl/fl (Rac1^fl/fl) mice 3 and 5 days post induction. Red arrow indicates disintegrating villus. Scale bar represents 100 μm in each case (See amplified images in Supplementary Fig. 1d–f). b Scanning EM on Vil-CreER^T2 (Wild-type) or Vil-CreER^T2 Rac1^fl/fl (Rac1^fl/fl) intestines 4 days post induction. Red arrows indicate rounded, blebbing cells. Scale bar represents 100 μm in the upper panels and 50 μm in the lower panels. c Vil-CreER^T2 driving expression of LifeAct-GFP in wild-type and Rac1^fl/fl intestines 4 days post induction. Scale bar represents 50 μm.

Fig. 2. VAV3 and TIAM1 are upregulated following APC loss.

a Heatmap derived from RNA-seq analysis comparing whole tissue from wild-type (Vil-CreER^T2) and APC intestines (Vil-CreER^T2 Apc^fl/fl) n = 3 biologically independent animals for both APC and WT intestinal tissue. Log₂FC of GEF expression displayed on the right, genes significantly deregulated displayed in bold (FDR < 0.05) displayed in bold. b RNAscope of Vav2 in the intestinal epithelium of Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl, Vav3^−/− (APC V3) mice, Scale bar represents 100 μm. c Quantification of Vav2 RNAscope from Vil-CreER^T2 Apc^fl/fl and Vil-CreER^T2 Apc^fl/fl Vav3^−/− intestines. N = 5 biologically independent animals for each genotype. P = 0.0079 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD.

Fig. 3. Loss of a single or double GEF is unable to rescue the Apc^fl/fl phenotype.

a H&E, BrdU incorporation and RNAscope for Lgr5 and Olfm4 on Vil-CreER^T2 Apc^fl/fl (APC), Vil-CreER^T2 Apc^fl/fl, Vav2^−/−, Vav3^−/− (APC V2V3) and Vil-CreER^T2 Apc^fl/fl Tiam1^−/− (APC T). Scale bar represents 100 μm for H&E and BrdU and 50 μm for RNAscope images. b Quantification of BrdU positive cells. N = 6, 5 and 6 biologically independent animals for Vil-CreER^T2 Apc^fl/fl (APC), Vil-CreER^T2 Apc^fl/fl, Vav2^−/−, Vav3^−/− (APC V2V3; p = 0. 0303 as determined by a two-tailed Mann–Whitney) and Vil-CreER^T2 Apc^fl/fl Tiam1^−/− (APC T; *p = 0.0260, as determined by a two-tailed Mann–Whitney) respectively. Data are presented as mean values ±SD. c Quantification of clonogenicity assay of intestinal organoids. N = 6, 3 and 3 biologically independent animals for Vil-CreER^T2 Apc^fl/fl (APC), Vil-CreER^T2 Apc^fl/fl, Vav2^−/−, Vav3^−/− (APC V2V3; p = 0.5476, as determined by a two-tailed Mann–Whitney) and Vil-CreER^T2 Apc^fl/fl Tiam1^−/− (APC T) respectively (p = 0.5476, as determined by a two-tailed Mann–Whitney). Data are presented as mean values ±SD. d, e Survival of Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (Lgr5 APC), Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/− (Lgr5 APC V2V3) and Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Tiam1^−/− (Lgr5 APC T). d N = 19 and 18 biologically independent animals for Lgr5 APC and Lgr5 APC V2V3 respectively (p = 0.7142, as determined by Log-rank (Mantel-Cox) test). e N = 15 and 10 biologically independent animals for Lgr5 APC and Lgr5 APC T respectively (under the control of Lgr5-EGFP-IRES-creER^T2). (p = 0.1982, as determined by Log-rank (Two-tailed Mantel-Cox test). The same Lgr5 APC control cohort was used in both d and e as well as in Fig. 4f and Supplementary Fig. 6D. f Quantification of intestinal tumour burden following APC loss in the Lgr5 stem cell compartment. Tumour burden is determined as percentage of intestine which is covered by lesion or adenomas. Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (APC; n = 9 biologically independent animals) vs Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/− (APC V2V3; n = 6 biologically independent animals) p = >0.9999 as determined by a two-tailed Kruskal–Wallis with Dunn’s multiple comparisons test. Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (APC) vs Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Tiam1^−/− (APC T; n = 7 biologically independent animals), p = 0.7532 as determined by a two-tailed Kruskal–Wallis with Dunn’s multiple comparisons test. Data are presented as mean values ±SD. g Solid adenomas (H&E) were observed in each genotype, indicated by the arrow. Scale bar represents 500 μm.

Fig. 4. Loss of three GEFs is able to suppress the loss of Apc phenotype.

a RNAscope staining for Vav2 in intestine from Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl Tiam1^−/− (APC T) intestines. Scale bar represents 100 μm. b Quantification of Vav2 RNAscope in Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl Tiam1^−/− (APC T) intestines. N = 5 biologically independent animals for each genotype *p = 0.0159 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD. c Images for H&E and BrdU incorporation and RNAscope for Lgr5 and Olfm4 on wild-type (WT), Vav2^−/−, Vav3^−/ Tiam1^−/− (V2V3T), Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl, Vav2^−/− and Vav3^−/−Tiam1^−/− (APC V2V3T). Scale bar represents 100 μm for H&E and BrdU and 50 μm for RNAscope images. d Quantification of BrdU positive cells. N = 7, 6 and 8 biologically independent animals for wild-type, Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl, Vav2^−/− and Vav3^−/− Tiam1^−/− (APC V2V3T) respectively. WT vs APC ***p = 0.0006, APC vs APC V2V3T *p = 0.0013 as determined by a one-tailed Mann–Whitney test. Control data (APC) as in Fig. 3c. Data are presented as mean values ±SD. e Quantification of clonogenicity assay of intestinal organoids. N = 6 biologically independent cell lines for both Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC V2V3T). *p = 0.0260 as determined by a two-tailed Mann–Whitney test. f Compared to Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (Lgr5 APC), Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/− Tiam1^−/− (Lgr5 APC V2V3T) mice have a significant survival advantage. N = 19 and 15 biologically independent animals for Lgr5 APC and Lgr5 APC V2V3T respectively. **p = 0.0091 as determined by a two-tailed Log-rank (Mantel-Cox) test. Lgr5 APC control cohort is the same cohort as used in Fig. 3d, e as well as Supplementary Fig. 6D. Data are presented as mean values ±SD. g Quantification of intestinal tumour burden at clinical endpoint following APC loss in the Lgr5 stem cell compartment. N = 8 and 9 biologically independent animals for Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (Lgr5 APC) and Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/− Tiam1^−/− (Lgr5 APC V2V3T) respectively. p = 0.9522 as determined by a two-tailed Mann–Whitney test. Data are presented as mean values ±SD. h Solid adenomas (asterisk) were observed in the Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl (Lgr5 APC) cohort, whereas cystic adenomas were observed (H&E) in Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/− Tiam1^−/− (Lgr5 APC V2V3T) and Lgr5-EGFP-IRES-creER^T2 Apc^fl/fl, Rac1^fl/fl (Lgr5 APC Rac1), as indicated by arrows. Scale bar represents 100 μm. i Intestinal tumour number in an AOM-DSS colitis-associated model of tumourigenesis. Study was carried out on wild-type mice or Vav2^−/−, Vav3^−/− Tiam1^−/− mice. N = 9 and 4 biologically independent animals for wild-type or Vav2^−/−, Vav3^−/− Tiam1^−/− respectively. **p = 0.0042 as determined by a one-tailed Mann–Whitney Test. Data are presented as mean values ±SD.

Fig. 5. Loss of the triple GEFs results in a downregulation of junctional RAC1 activity.

a Immunoprecipitation of Active RAC1 from intestinal epithelium, blotted with total RAC1 and the corresponding scoring. n = 3 biologically independent samples for Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC V2V3T). *p = 0.00799 as determined by a one-tailed Mann–Whitney test. Data are presented as mean values ±SD. b FLIM-FRET analysis of organoids day 3 post isolation from Vil-CreER^T2 Apc^fl/fl (APC) and Vil-CreER^T2 Apc^fl/fl Vav2^−/−, Vav3^−/−, Tiam1^−/− (APC V2V3T) Scale bar represents 20 μm. c Quantification of FLIM-FRET analysis at cell–cell contacts. N = 3 biologically independent cell lines derived from independent mice for each genotype. *p = 0.0426 as determined a two-tailed unpaired T-test. Data are presented as mean values ±SD.

Fig. 6. Oncogenic mutation of KRAS drives resistance to RAC-GEF depletion.

a Quantification of BrdU positive cells. N = 5, 5 and 10 biologically independent animals for Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS), Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) and Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/−, and Vav3^−/− Tiam1^−/− (APC KRAS V2V3T) respectively. APC KRAS vs APC KRAS Rac1^fl/fl p = 0.0461 APC KRAS vs APC KRAS V2V3 p = 0.9363 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreER^T2). Data are presented as mean values ±SD. b Representative images for BrdU incorporation of Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS), Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRas Rac1^fl/fl) and Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/− and Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). Scale bar represents 100 µm. c Representative images of RNAscope for Lgr5 and Olfn4 and IHC for CD44 and SOX9 Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS), Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) and Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/− and Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). Scale bar represents 100 µm. d Quantification of Lgr5 RNAscope. Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) p = 0.1105 APC KRAS vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). n = 6, 5 and 8 biologically independent animals for APC KRAS, APC KRAS Rac1^fl/fl, and APC KRAS V2V3T respectively. p = >0.999 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreER^T2). Data are presented as mean values ±SD. e Quantification of Olfm4 RNAscope. Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS) vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) p = 0.356 APC KRAS vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). n = 6, 4 and 9 biologically independent animals for APC KRAS, APC KRAS Rac1^fl/fl and APC KRAS V2V3T respectively. p = 0.9381 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreER^T2). Data are presented as mean values ±SD. f Quantification of CD44 IHC. Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS) vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) p = 0.2061 APC KRAS vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). n = 7, 4 and 8 biologically independent animals for APC KRAS, APC KRAS Rac1^fl/fl and APC KRAS V2V3T respectively. p = >0.999 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreER^T2). Data are presented as mean values ±SD. g Quantification of SOX9 IHC. Vil-CreER^T2 Apc^fl/fl KRas^G12D (APC KRAS) vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Rac1^fl/fl (APC KRAS Rac1^fl/fl) p = >0.999 APC KRAS vs Vil-CreER^T2 Apc^fl/fl, KRas^G12D, Vav2^−/−, Vav3^−/− Tiam1^−/− (APC KRAS V2V3T). n = 7, 5 and 9 biologically independent animals for APC KRAS, APC KRAS Rac1^fl/fl and APC KRAS V2V3T respectively p = 0.4088 as determined by a two-tailed Kruskal–Wallis statistical analysis with Dunn’s mutltiple comparisons test (under the control of Vil-CreER^T2). Data are presented as mean values ±SD.