CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss

Abstract

NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1^S186A/-, Nkx3.1^+/- , and Nkx3.1^+/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1^S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1^+/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.

Figure 1.

Prostate lobe weights at different time points. Statistical differences between Nkx3.1^+/− mice with Nkx3.1^+/+ and Nkx3.1^S186A/- mutant mice was analyzed by t-test. * p<0.05.

Figure 2.

Histologic sections of anterior prostate lobe at different time points. H&E staining of anterior prostate sections are shown for each genotype at 2, 6, and 10 months of age. The higher magnifications show paucicellular areas seen in glands of Nkx3.1^S186A/- mice but not in other genotypes.

Figure 3.

Nkx3.1 expression. Nkx3.1 was detected in paraffin sections from anterior prostate lobes of Nkx3.1^+/+, Nkx3.1^+/−, and Nkx3.1^S186A/- mice at 2, 6, and 10 months of age. Counterstain was done with antibody to β-catenin and DAPI. Confocal images were quantitated with image J and Nkx3.1 expression was plotted relative to adjacent β-catenin expression. Data are from a minimum of ten fields. Nkx3.1 expression in Nkx3.1^S186A/- mice differed from both Nkx3.1^+/+ and Nkx3.1^+/− mice by t-test with p<0.01.

Figure 4.

Cell proliferation. Immunostaining for Ki-67 was done with cytokeratin 8 and DAPI as counterstains in sections of anterior prostate lobes. Confocal images were quantitated with image J and the number of Ki-67-positive cells per field as a percentage of all nuclei in the field were recorded. A minimum of ten fields were assayed and all cells were counted. Each point represents the percentage of Ki-67 positive cells in one field. The distribution of Ki-67 positive cells in Nkx3.1^+/− mice differed from both Nkx3.1^+/+ and Nkx3.1^S186A/- mice at both 6 and 10 months by t-test with p<0.01.

Figure 5.

TUNEL staining was done on paraffin sections from anterior prostate lobes of Nkx3.1^+/+, Nkx3.1^+/−, and Nkx3.1^S186A/- mice at 2, 6, and 10 months of age. Counterstain was done with DAPI. Confocal images were quantitated with image J and the number of TUNEL-positive cells per field were recorded compared to the total number of nuclei seen in the field. A minimum of ten fields were assayed and each point represents the percentage of TUNEL positive cells in a field. The percentage of TUNEL positive cells in Nkx3.1^S186A/- mice differed from both Nkx3.1^+/+ and Nkx3.1^+/− mice by t-test with p<0.01.

Figure 6.

Metabolic and Stress Response Activation Suppressed by Nkx3.1(S186A). The figure shows confocal immunofluorescent micrographs of anterior prostate lobes from 2-month old mice of the indicated genotype. The micrographs on the far right show the staining of prostates from wild-type mice 2 hours after exposure to 15Gy γ-irradiation. Cytokeratin 5 was employed for pS6 and nitrotyrosine staining to show preservation of histologic architecture. Staining intensities were measured by image J and shown relative to intensity in Nkx3.1^+/+ mice in the respective histograms on the far right.