Nkx3.1 and Myc crossregulate shared target genes in mouse and human prostate tumorigenesis

Abstract

Cooperativity between oncogenic mutations is recognized as a fundamental feature of malignant transformation, and it may be mediated by synergistic regulation of the expression of pro- and antitumorigenic target genes. However, the mechanisms by which oncogenes and tumor suppressors coregulate downstream targets and pathways remain largely unknown. Here, we used ChIP coupled to massively parallel sequencing (ChIP-seq) and gene expression profiling in mouse prostates to identify direct targets of the tumor suppressor Nkx3.1. Further analysis indicated that a substantial fraction of Nkx3.1 target genes are also direct targets of the oncoprotein Myc. We also showed that Nkx3.1 and Myc bound to and crossregulated shared target genes in mouse and human prostate epithelial cells and that Nkx3.1 could oppose the transcriptional activity of Myc. Furthermore, loss of Nkx3.1 cooperated with concurrent overexpression of Myc to promote prostate cancer in transgenic mice. In human prostate cancer patients, dysregulation of shared NKX3.1/MYC target genes was associated with disease relapse. Our results indicate that NKX3.1 and MYC coregulate prostate tumorigenesis by converging on, and crossregulating, a common set of target genes. We propose that coregulation of target gene expression by oncogenic/tumor suppressor transcription factors may represent a general mechanism underlying the cooperativity of oncogenic mutations during tumorigenesis.

Figure 1. Analysis of genome-wide Nkx3.1-binding sites in the mouse prostate.

(A) Visualization of Nkx3.1-binding sites across all mouse chromosomes in Nkx3.1^+/+ (WT), Nkx3.1^+/– (HET), and Nkx3.1^–/– (KO) mouse prostates. Not to scale. (B) High reproducibility in Nkx3.1-binding sites among biological replicates and genotypes. Chromosome 1 is shown as an example. (C) Integrated Genome Browser (IGB) shots showing Nkx3.1-binding sites near the Pbsn and Ar genes. (D) Euler diagram showing overlap of Nkx3.1-binding sites in WT and HET mouse prostates. See also Supplemental Figure 1 and Supplemental Table 1.

Figure 2. Analysis of the Nkx3.1 cistrome.

(A) Spatial distribution of Nkx3.1 binding sites genome wide (red) or near genes as shown in the RefSeqGene database ( http://www.ncbi.nlm.nih.gov/refseq/rsg/) (orange). The results from 1 WT prostate and 1 HET prostate are shown. (B) Enrichment of Nkx3.1-binding sites approximately 150 nt upstream of the TSS for all Nkx3.1-positive samples. Shown are results for MACS peak calling algorithm with the P value set at the default 10^–5 and at 10^–10. (C) The number of times Nkx3.1 binds target genes is indicated. bs, binding site. (D) Top panel: in vitro defined Nkx3.1 motif (29). Bottom panel: consensus motif for Nkx3.1, as determined by querying the 1,000 most enriched ChIP-seq loci with MEME (v. 4.6.0). See also Supplemental Tables 2 and 3.

Figure 3. Identification of direct Nkx3.1 target genes.

(A) GSEA analysis of genes whose expression is dysregulated in Nkx3.1^–/– mice identifies significant downregulation of genes involved in aminoacyl tRNA synthesis and enrichment of genes involved in the cell cycle and oxidative stress in the KO mouse prostate. (B) Integration of Nkx3.1 ChIP-seq data with microarray analysis of Nkx3.1^–/– mice identifies 282 direct Nkx3.1 target genes. (C) Venn diagrams showing the fraction of genes activated or repressed by Nkx3.1 that are also bound by Nkx3.1 by ChIP-seq, i.e., direct activated genes and direct repressed genes. (D) GO analysis of direct Nkx3.1 target genes by WebGestalt. See also Supplemental Figure 2 and Supplemental Table 4.

Figure 4. Identification of a subset of direct Nkx3.1 target genes coregulated by Myc.

(A) Network analysis using GeneGO MetaCore software identifies a subset of direct Nkx3.1 target genes that are known to be bound by Myc (P = 3.94 × 10^–169). Genes upregulated in the Nkx3.1 KO prostates are shown as blue circles, while those downregulated are indicated as red circles in the diagram. (B) Relative locations of actual Nkx3.1- and Myc-binding sites in selected Nkx3.1/Myc coregulated genes identified from genome-wide binding studies. (C) ChIP-PCR validation of Myc binding to selected shared Nkx3.1/Myc target genes in Myc-CaP mouse prostate adenocarcinoma cell line (top). These cells express Myc but not Nkx3.1, as shown in the inset Western blot. Results are representative of at least 2 independent experiments. (D) ChIP-qPCR validation of MYC and NKX3.1 binding to selected shared NKX3.1/MYC target genes in LNCaP human prostate adenocarcinoma cell line. These cells express both MYC and NKX3.1, as shown in the inset Western blot. Results are presented as mean ± sd from at least 2 independent experiments. See also Supplemental Tables 5 and 6.

Figure 5. NKX3.1 and Myc interact and coregulate expression of shared target gene HK2.

(A) Myc and NKX3.1 coimmunoprecipitation. Cell lysates from 293T cells expressing HA-NKX3.1 and Flag-Myc were immunoprecipitated with the indicated antibodies and immunoblotted for HA. To deplete DNA, ethidium bromide (EtBr) was added to lysates for 30 minutes prior to immunoprecipitation and during washes. (B) Coimmunoprecipitation of HA-NKX3.1 and Flag-Myc WT (lane 2), Flag-Myc mutant 1 lacking Myc–box 1 (lane 3), or Flag-Myc mutant 2 lacking Myc–box 2 (lane 4) in 293 cells. Asterisk indicates a nonspecific band. (C) ChIP–re-ChIP assay. LNCaP cells were subjected to ChIP by NKX3.1 antibody followed by ChIP with Myc antibody or the reverse and binding interrogated at the HK2 promoter E box. (D) Depletion of NKX3.1 by siRNA in LNCaP cells leads to upregulation of HK2 expression compared with control siLuc (luciferase siRNA) treatment. (E) Depletion of NKX3.1 by siRNA in LNCaP-Myc ER cells enhances activation of HK2 gene expression by tamoxifen (OHT) compared with control siGFP treatment (left panel). In contrast, expression of exogenous Nkx3.1 inhibited OHT induction of Hk2 in DKO cells. Results are representative of at least 2 independent experiments. Error bars represent mean ± SEM. See also Supplemental Figure 4.

Figure 6. Dysregulation of shared Nkx3.1/MYC target genes in mouse and human prostate cancer tissues.

(A) Dysregulation of Nkx3.1/MYC targets in a prostate tissue recombination model. Affymetrix arrays were used to compare gene expression between prostate grafts regenerated with mouse prostate epithelial cells transduced with lentivirus overexpressing human MYC or control lentivirus. The MYC grafts contained HGPIN lesions, overexpressed MYC, and downregulated Nkx3.1 (see Supplemental Figure 3). By significance analysis of microarrays (SAM) and employing a 1.4-fold cutoff, 20 of the 65 Nkx3.1/MYC target genes were significantly altered in the grafts containing HGPIN lesions with MYC overexpression and Nkx3.1 downregulation. (B) GSEA analysis shows association between expression of NKX3.1/MYC target genes and relapse in human prostate tumors. Gene expression arrays from tumors consisting of 32 tumors with relapse and 34 tumors without relapse were analyzed. See also Supplemental Figures 3 and 5, and Supplemental Tables 5–7.

Figure 7. Loss of Nkx3.1 facilitates MYC-initiated prostate tumorigenesis in transgenic mice.

(A) Scheme for concurrent overexpression of MYC and deletion of Nkx3.1 in the prostates of transgenic mice. The Z-MYC construct contains a latent human MYC allele under the control of the CMV enhancer/actin promoter. The floxed allele of Nkx3.1 has loxP sites flanking exon 2, which encodes the Nkx3.1 homeodomain. Cre recombinase is under the control of the prostate-specific Probasin promoter (PB-Cre). When Cre is expressed, the MYC gene will be overexpressed and the Nkx3.1 gene will be concurrently deleted. (B) Graph summarizing prostate histology of Nkx3.1/MYC mutant mice. *P < 0.01 for PBCre;Z-MYC;Nkx3.1^f/f relative to PBCre;Z-MYC. (C) Histological characterization of prostate lesions in Nkx3.1/MYC mutant mice. H&E-stained sections of 35-week-old mouse prostates. Note high-grade PIN lesions in PBCre;Z-MYC;Nkx3.1^f/+ and PBCre;Z-MYC;Nkx3.1^f/f mice. Insets show higher magnifications (×40). Arrows denote mitotic figures. Scale bars: 50 μm. (D) Proliferation in mutant mice prostates assessed by phospho-histone H3 staining. *P < 0.05. Error bars represent mean ± SEM. (E) Areas of focal loss of SMA (arrows) in 35-week-old PBCre;Z-MYC;Nkx3.1^f/f mice, suggestive of microinvasion. Scale bar: 50 μm. See also Supplemental Figures 6 and 7.

Figure 8. Nkx3.1 and MYC coregulate prostate tumorigenesis and target gene expression.

(A) Scheme for prostate regeneration by tissue recombination to recapitulate phenotypes of transgenic mice. Pieces of transgenic mouse prostates were combined with rat UGM and collagen and then implanted orthotopically into prostates of SCID mice. Ten weeks later, regenerated prostates were harvested. (B) Graph summarizing pathology of regenerated prostates. *P < 0.05 for PBCre;Z-MYC;Nkx3.1^f/f relative to all others. (C) Histological characterization of prostate lesions in regenerated prostates. H&E-stained sections of 10-week-old grafts. PBCre;Z-MYC and PBCre;Nkx3.1^f/f grafts show mostly normal glands with areas of hyperplasia and dysplasia consistent with low-grade PIN, while PBCre;Z-MYC;Nkx3.1^f/f prostates show multiple foci of HGPIN. Scale bars: 50 μm. (D) Focal loss of SMA (arrows) consistent with microinvasion in 10-week-old PBCre;Z-MYC;Nkx3.1^f/f prostate grafts. Asterisks indicate benign glands. Scale bars: 50 μm. (E–G) Upregulation of Nedd4l and Hk2 and downregulation of Prdx6 expression by immunohistochemistry in HGPIN/cancer lesions from 10-week-old PBCre;Z-MYC;Nkx3.1^f/f prostate grafts (arrows). Note that the sections in E are adjacent to those in D. Asterisks indicate benign glands. Scale bars: 50 μm. See also Supplemental Figures 7 and 8.

Figure 9. Model for crossregulation of prostate tumorigenesis by convergence of NKX3.1 and MYC on shared target genes.

NKX3.1 and MYC protein levels vary during tumorigenesis, ranging from low MYC/high NKX3.1 in benign tissue to high MYC/low NKX3.1 in more advanced tumors. In benign tissue, where NKX3.1 protein levels are high and MYC levels are very low, shared target genes such as HK2 are bound and repressed by NKX3.1. In samples in which both NKX3.1 and MYC are expressed, NKX3.1 can bind to its consensus DNA site as well as form a complex with MYC to dampen MYC’s transcriptional activity and target gene expression. In advanced tumors, where MYC is highly expressed and NKX3.1 expression lost, MYC binds and activates its target genes unopposed. Thus, as tumorigenesis progresses, the MYC:NKX3.1 ratio increases and the expression of protumorigenic shared target genes (activated by MYC and repressed by NKX3.1) increases. In the model depicted here, we show NKX3.1 as a transcriptional repressor and MYC as an activator of protumorigenic target genes. However, the converse, where NKX3.1 activates and MYC represses antitumorigenic target genes, also fits the general model. See also Supplemental Figure 9.