Supplementary Tables 1-4: Table 1. Primers for PCR amplification for CRISPR/Cas9 mouse models. Table 2. ssODN oligos. Table 3. Primers for genotyping of mouse mutation. Table 4. Antibodies used for immunofluorescence staining. Supplemental Figures 1-8: Supplementary Figure 1 A. Amino acid sequences of human (top) and murine (bottom) NKX3.1 showing consensus (central) sequence and location of serine 185 and 186 indicated by the box in the figure. B. Effect of S186A mutation on half-life of murine Nkx3.1. Supplementary Figure 2 A. Schematic of targeted sites for mutation at locus. Supplementary Fig 3 A. H&E staining of dorsolateral prostatic lobes at different time points. Results similar to those seen with anterior prostates are shown in the figure. Supplementary Fig 4 A. Immunohistochemistry for cytokeratins 5 and 8 in 2-month old anterior prostate lobes. The arrow in the section from Nkx3.1 show an area of early hyperplasia. The arrows in the section from Nkx3.1 show areas of epithelial detachment. Supplementary Figure 5 Nkx3.1 expression. Supplementary Fig 6 Cell proliferation. Supplementary Figure 7 TUNEL staining was done on paraffin sections from dorsolateral prostate lobes of Nkx3.1 , Nkx3.1 , and Nkx3.1 mice at 2, 6, and 10 months of age. Supplementary Figure 8 A. Cleaved caspase 3 staining was done on paraffin sections from both anterior and dorsolateral prostate lobes of Nkx3.1 , Nkx3.1 , and Nkx3.1 mice at 2, 6, and 10 months of age.
ARTICLE 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. Nkx3.1S186A/−, 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 Nkx3.1S186A/− 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.
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.