Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner

Abstract

WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1^+/- mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1-p53 loop may be targeted to manage inflammatory diseases and cancer.

Keywords: Ninjurin 1; cell adhesion; inflammation; mutant p53; p53.

Fig. 1.

Ninj1 regulates WT and mutant p53 expression. (A–C) Ninj1, p53, Egr-1, and actin proteins were examined by Western blotting in MCF7 (A), SW480 (B), and MIA-PaCa2 (C) cells transiently transfected with a control or Ninj1-expressing vector. (D–F) Ninj1, p53, Egr-1, and actin proteins were examined by Western blotting in isogenic controls and Ninj1-KO MCF7 (D), SW480 (E), and MIA-PaCa2 (F) cells.

Fig. 2.

The effect of Ninj1 on cell growth and migration is p53-dependent. (A–C) Colony formation assay was performed with isogenic controls, Ninj1-KO MCF7 (A), SW480 (B), and MIA-PaCa2 (C) cells. (D–F) Wound-healing assay was performed with isogenic controls and Ninj1-KO MCF7 (D), SW480 (E), and MIA-PaCa2 (F) cells. The images were proportionally scaled with an original magnification of 100×.

Fig. 3.

Mice deficient in Ninj1 are prone to systemic inflammation. (A) Kaplan–Meier survival curve for WT vs. Ninj1^+/− mice (P = 0.080 by log-rank test). (B) The percentage of mice with dermatitis in WT vs. Ninj1^+/− mice (P = 0.0192 by Fisher’s exact test). (C) Representative images of H&E-stained skin sections from a pair of gender-matched WT and Ninj1^+/− mice. (D) Representative images of H&E- and IHC-stained liver and kidney sections from a pair of gender-matched WT and Ninj1^+/− mice. The red arrows indicate the positive-staining cells. The images were proportionally scaled with an original magnification of 200×.(E and F) IL-6 and actin transcripts were examined in the liver and kidney of two pairs of gender-matched WT and Ninj1^+/− mice. WT#1: F/56w/ID#5-19-3. WT#2: F/50w/ID#9-3-2. Ninj1^+/− #1: F/51w/ID#11-8-5. Ninj1^+/− #2: F/44w/ID#12-2-4.

Fig. 4.

Mice deficient in Ninj1 are prone to insulitis. (A) Representative images of H&E-stained pancreas sections from two pairs of gender-matched WT and Ninj1^+/− mice. The images were proportionally scaled with an original magnification of 200×. (B) The incidence of insulitis in WT vs. Ninj1^+/− mice (P = 0.0088 by Fisher’s exact test). (C and D) Pancreas sections from WT (C) and Ninj1^+/− (D) mouse were stained with antibodies against CD3, B220, or F4/80. The box plots were used to show the staining area in the Right. Red arrow: T cells at the inflammation site. Blue arrow: T cells inside the islet. Yellow arrow: B cells at the inflammation site.

Fig. 5.

Ninj1 deficiency shortens the lifespan, increases the tumor burden, and alters the tumor spectrum in p53^R270H/− mice. (A) Kaplan–Meier curve for p53^R270H/− vs. Ninj1^+/−;p53^R270H/− mice (P = 0.014 by log-rank test). (B) Tumor spectrum in p53^R270H/− vs. Ninj1^+/−;p53^R270H/− mice. (C) The incidence of T-LBL in p53^R270H/− vs. Ninj1^+/−;p53^R270H/− mice (P = 0.0029 by Fisher’s exact test). (D) Representative images of H&E-stained T-LBL disseminated to peripheral organs in a Ninj1^+/−;p53^R270H/− mouse. (E) The incidence of disseminated T-LBL in p53^R270H/− vs. Ninj1^+/−;p53^R270H/− mice (P = 0.0007 by Fisher’s exact test). (F) The tumor burden in p53^R270H/− vs. Ninj1^+/−;p53^R270H/− mice (P = 0.0039 by Fisher’s exact test). (G) Representative images of H&E- and IHC- (p53) stained kidney sections from normal and sarcoma areas in Ninj1^+/−;p53^R270H/− mouse. (H) p53 and actin protiens were examined in T-LBLs from two pairs of p53^R270H/− and Ninj1^+/−;p53^R270H/− mice. T-LBL#1: p53^R270H/−/M/16w/ID#1-15-4. T-LBL#2: Ninj1^+/−;p53^R270H/−/M/15w/ID#2-28-2. T-LBL#3: p53^R270H/−/M/18w/ID#11-26-18. T-LBL#4: Ninj1^+/−;p53^R270H/−/M/18w/ID#11-5-7.

Fig. 6.

Ninj1 deficiency increases the penetrance of T-LBL in p53^−/− mice. (A) Kaplan–Meier curve for p53^−/− vs. Ninj1^+/−;p53^−/− mice (P = 0.669 by log-rank test). (B) The incidence of T-LBL in p53^−/− vs. Ninj1^+/−;p53^−/− mice (P = 0.0054 by Fisher’s exact test). (C) Tumor spectrum in p53^−/− vs. Ninj1^+/−;p53^−/− mice.