Targeting p53-dependent stem cell loss for intestinal chemoprotection

Abstract

The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type-specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5⁺ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.

Fig. 1. PUMA mediates CPT-11–induced intestinal injury

Mice with indicated genotypes were treated with CPT-11 (200 mg/kg) once or as specified and analyzed at indicated times. (A) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining in intestinal crypts at 6 hours. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei. Scale bar, 50 μm. (B) Quantitation of TUNEL⁺ columnar cells at the crypt bottom (CBCs) and +4 to 9 cells in (A). (C) Expression of indicated proteins was analyzed by Western blotting at 6 hours. Lysates were pooled from the intestinal mucosa of three mice. Representative results are shown, and similar results were obtained in at least three independent experiments. (D) Puma RNA in situ hybridization (ISH) in the crypts at 6 hours. Scale bar, 20 μm. In (A) and (D), arrows indicate CBCs at positions 1 to 3 below Paneth cells, and asterisks indicate +4 to 9 cells at positions 4 to 9 above the CBCs. In (A) to (D), n = 3 mice per group. In (B), values are means + SEM. ***P < 0.001 (two-tailed Student’s t test), knockout (KO) versus wild type (WT). (E) Survival of mice treated with three consecutive daily doses of CPT-11 (215 mg/kg per day) on days 0, 1, and 2. WT versus Puma KO, P = 0.0004; WT versus p53 KO, P = 0.382 (log-rank test). (F) Hematoxylin and eosin (H&E) staining of the small intestine from mice on day 5 treated as in (E). Scale bar, 200 μm.

Fig. 2. PUMAi protects against CPT-11–induced intestinal injury

Mice were treated with CPT-11 with or without PUMA inhibitor (PUMAi). PUMAi (10 mg/kg) or vehicle was given intraperitoneally for 2 hours before CPT-11 or as specified. The small intestine or indicated tissue was analyzed at the indicated times. (A) Representative images of TUNEL staining in intestinal crypts of mice treated with CPT-11 (200 mg/kg). DAPI was used to stain nuclei. Scale bar, 50 μm. Arrows indicate CBCs, and asterisks indicate +4 to 9 cells. (B) Quantitation of TUNEL⁺ CBCs and transit-amplifying cells from (A). Inh, inhibitor. (C) Tissue distribution of PUMAi at different times after a single injection. (D) Quantitation of TUNEL⁺ crypt cells in mice 6 hours after CPT-11 (200 mg/kg) treatment. PUMAi (10 mg/kg) was given 2 hours before or 1 hour after CPT-11 treatment. n = 3 mice per group (B to D). In (B) and (D), values are means + SEM. *P < 0.05 (two-tailed Student’s t test). Veh, vehicle. (E) Survival of WT mice after three doses of CPT-11 (215 mg/kg per day) (done with the control in Fig. 1E). PUMAi (10 mg/kg) was given 2 hours before each dose of CPT-11. P = 0.0047 (log-rank test). (F) H&E staining of the small intestine from mice treated as in (E) on days 0, 5 and 30. Scale bar, 200 μm.

Fig. 3. PUMA deficiency protects tumor-bearing mice from chemotherapy-induced GI injury

WT and Puma KO mice bearing Lewis lung carcinoma (LLC) tumors were treated with CPT-11 (200 mg/kg) six times over 15 days, on days 11, 14, 17, 19, 23, and 25. Mice or tumors were analyzed at the indicated times. (A) Tumor volumes were measured every other day from days 11 to 25. CT, vehicle control. (B) Body mass was expressed as a percentage of that on day 11 for each mouse from days 11 to 25. (C) H&E staining of the small intestine tissue on day 25. Scale bar, 100 μm. (D) Villus height from (C). Measurements were from a minimum of 40 villi per mouse. (E) Quantitation of intestinal crypts per field from (C). (F) Intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used to stain nuclei. Scale bar, 100 μm. (G) Quantitation of neutrophils per 400× field from the same slides as (F). In (A), (B), (D), (E), and (G), values are means + SEM; n = indicated (A and B) or 3 to 4 mice per group (D, E, and G). **P < 0.01 and ***P < 0.001 (two-tailed Student’s t test). Puma KO, CPT versus control (A); CPT-11–treated, Puma KO versus WT (B).

Fig. 4. PUMAi protects tumor-bearing mice from chemotherapy-induced GI injury

WT mice bearing LLC tumors were treated with CPT-11 (200 mg/kg) six times over 15 days on days 11, 14, 17, 19, 23, and 25. Vehicle or PUMAi was given 2 hours before and 20 hours after each dose of CPT-11. Mice or tumors were analyzed at the indicated times. (A) Tumor volumes were measured starting on day 11. (B) Body mass was expressed as a percentage of that on day 11 for each mouse from days 11 to 25. (C) H&E staining of the small intestine tissue on day 25. Scale bar, 100 μm. (D) Villus height from (C). Measurements were from a minimum of 40 villi per mouse. (E) Quantitation of intestinal crypts per field from (C). (F) Intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used to stain the nuclei. Scale bar, 100 μm. (G) Quantitation of neutrophils per 400× field from the same slides as (F). In (A), (B), (D), (E), and (G), values are means + SEM; n = indicated (A and B) or 3 to 4 mice per group (D, E, and G). *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t test). PUMAi, CPT versus control (A); CPT-11–treated mice, PUMAi versus CT (B).

Fig. 5. Targeting PUMA protects the LGR5⁺ stem cells against CPT-11

WT and Puma KO mice were treated with CPT-11 (200 mg/kg) and analyzed at the indicated times. PUMAi (10 mg/kg) was given once 2 hours before CPT-11 treatment. (A) Green fluorescent protein (GFP) (LGR5) and TUNEL immunofluorescence staining in intestinal crypts. Scale bar, 50 μm. Arrow indicates double positive cell. (B) Quantitation of GFP/TUNEL–double-positive cells per GFP-positive crypt from (A). (C) Immunofluorescence staining for CD166 in the intestinal crypts. Scale bar, 50 μm. (D) Quantitation of CD166 cells in the +4 to 9 region in crypts. (E) Indicated mRNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Complementary DNAs (cDNAs) were synthesized from RNA pooled from three mice. Values were normalized to Gapdh expression and expressed relative to each gene’s own 0-hour control. (F) 53BP1 immunofluorescence staining in intestinal crypts. Scale bar, 50 μm. (G) Quantitation of 53BP1⁺ crypt cells at 6 and 48 hours after single CPT-11 treatment or #120 hours with three daily doses of CPT-11 (215 mg/kg per day) as in Fig. 2E. In (C) and (F), arrows indicate CBCs, and asterisks indicate +4 to 9 cells. In (A), (C), and (F), DAPI was used to stain the nuclei. In (B), (E), and (G), values are means + SEM; n = 3 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001 [one-way analysis of variance (ANOVA) with Tukey post hoc test performed separately for each time point]. In (D), values are means + SEM; n = 3 mice per group. **P < 0.01 (two-tailed Student’s t test).

Fig. 6. Targeting PUMA prevents LGR5⁺ stem cell exhaustion after repeated CPT-11 exposure

WT and Puma KO mice carrying the Lgr5-EGFP-IRES-creER^T2 marking allele were treated with CPT-11 and PUMAi over 2 weeks as in Fig. 5 and analyzed for intestinal phenotypes. (A) GFP (LGR5) immunofluorescence in intestinal crypts. Arrows indicate LGR5⁺ (GFP) crypts. Scale bar, 100 μm. (B) Quantitation of intestinal crypts containing at least one GFP⁺ (LGR5⁺) cell. (C) Top: Olfm4 RNA ISH in intestinal crypts. Scale bar, 50 μm. Asterisks indicate Olfm4⁺ crypt cells. Bottom: Percentage of crypts containing at least one Olfm4⁺ cell. (D) MMP7 immunofluorescence in intestinal crypts. Scale bar, 100 μm. (E) Quantitation of MMP7⁺ crypt cells at the crypt base (CBC region). (F) Top: GFP (LGR5) and MMP7 immunofluorescence in intestinal crypts. Scale bar, 20 μm. Asterisks indicate LGR5⁺/MMP7⁺ cell pairs. Bottom: Quantitation of LGR5⁺/MMP7⁺ cell pairs per crypt. In (A), (D), and (F), DAPI was used to stain nuclei. In (B), (C), (E), and (F), values are means + SEM; n = 3 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Tukey post hoc test).

Fig. 7. PUMAi protects mouse and human colonic culture against CPT-induced damage

(A) Representative images of mouse colonic organoids 6 days after CPT treatment. Organoids were treated with vehicle control (CT) or 500 nM CPT (day 1) for 24 hours with or without 50 μM PUMAi and monitored for growth until day 7. Scale bar, 500 μm. (B) Quantitation of organoids 100 μm or greater in diameter per field from (A). (C) Western blots for active (cleaved) caspase-3 (Casp3) from organoids 24 hours after CPT treatment. (D) Indicated mouse mRNAs were analyzed by qRT-PCR 24 hours after CPT treatment. NS, not significant. (E) Representative images of human colonic organoids treated as in (A). Scale bar, 500 μm. (F) Quantitation of organoids 100 μm or greater in diameter per field from (E). (G) Western blots for active (cleaved) caspase-3 from human organoids 24 hours after CPT treatment. (H) Indicated human mRNAs analyzed 24 hours after CPT treatment. In (C) and (G), lysates were prepared from three wells. Actin was used as a control for protein loading. In (D) and (H), cDNAs were synthesized from RNA pooled from three cultured wells. Values were normalized to Gapdh and expressed relative to vehicle controls. In (B), (D), (F), and (H), values are means + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Tukey post hoc test).