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Identification of an NKX3.1-G9a-UTY transcriptional regulatory network that controls prostate differentiation

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Science  24 Jun 2016:
Vol. 352, Issue 6293, pp. 1576-1580
DOI: 10.1126/science.aad9512

Clues to cancer from an identity change

The prostate and seminal vesicle have closely related developmental histories and both are regulated by the same androgenic hormones. A better understanding of the molecular mechanisms controlling the development of the two tissues could help solve why cancer arises frequently in the prostate but only rarely in seminal vesicles. Working with cell and mouse models, Dutta et al. show that forced expression of a single gene, the homeobox gene NKX3.1, causes seminal vesicle epithelium to differentiate into prostate. NKX3.1 regulates the expression of a gene program associated with prostate differentiation by interacting with the G9a histone methyltransferase. Disruption of this regulatory network probably contributes to prostate cancer development.

Science, this issue p. 1576

Abstract

The NKX3.1 homeobox gene plays essential roles in prostate differentiation and prostate cancer. We show that loss of function of Nkx3.1 in mouse prostate results in down-regulation of genes that are essential for prostate differentiation, as well as up-regulation of genes that are not normally expressed in prostate. Conversely, gain of function of Nkx3.1 in an otherwise fully differentiated nonprostatic mouse epithelium (seminal vesicle) is sufficient for respecification to prostate in renal grafts in vivo. In human prostate cells, these activities require the interaction of NKX3.1 with the G9a histone methyltransferase via the homeodomain and are mediated by activation of target genes such as UTY (KDM6c), the male-specific paralog of UTX (KDM6a). We propose that an NKX3.1-G9a-UTY transcriptional regulatory network is essential for prostate differentiation, and we speculate that disruption of such a network predisposes to prostate cancer.

Among the tissues of the male urogenital system, the prostate and seminal vesicle are secretory organs that develop in close proximity under the influence of androgens (fig. S1A) (1, 2). However, the prostate develops from the urogenital sinus, an endodermal derivative, whereas the seminal vesicle develops from the Wolffian duct, a mesodermal derivative. Among genes that distinguish prostate and seminal vesicle, the Nkx3.1 homeobox gene is among the earliest expressed in the presumptive prostatic epithelium during development, and its expression in adults is primarily restricted to prostatic luminal cells (3, 4), which are secretory cells that are the major target of prostate neoplasia (4, 5). Accordingly, in mouse models, loss of function of Nkx3.1 results in impaired prostate differentiation and defects in luminal stem cells, as well as predisposes to prostate cancer (3, 4).

Analyses of expression profiles from Nkx3.1 wild-type (Nkx3.1+/+) and Nkx3.1 mutant (Nkx3.1–/–) prostates revealed down-regulation of genes associated with prostate differentiation, such as FoxA1 (Forkhead Box A1), Pbsn (Probasin), HoxB13, and Tmprss2 (Transmembrane Protease, Serine 2), as well as luminal cells (cytokeratins 8 and 18), and up-regulation of basal cell markers (cytokeratins 5 and p63) (Fig. 1A, fig. S2A, and database S1) (6). Surprisingly, Nkx3.1–/– versus Nkx3.1+/+ prostates display up-regulation of genes that are expressed, albeit not exclusively, in seminal vesicle, namely Svs6, Sva, Svs4, and Svs5 (Fig. 1A and fig. S2A) (7). These differentially expressed genes were significantly enriched in a gene signature comparing seminal vesicle versus normal prostate, and this pattern was conserved in mouse and humans (Fig. 1B and figs. S1B and S2B). At the cellular level, we observed reduced expression of Probasin and a corresponding up-regulation of Svp2 in Nkx3.1–/– versus Nkx3.1+/+ prostates (figs. S1, C to E, and S2C; and tables S1 to S3) (8).

Fig. 1 Murine Nkx3.1 respecifies a nonprostatic epithelium to form prostate in vivo.

(A) Heat map representations of differentially expressed genes from Nkx3.1+/+ and Nkx3.1−/− prostate (6). (B) Gene set enrichment analysis, using as the query gene set differentially expressed genes from seminal vesicle versus prostate, compared with a reference gene signature of Nkx3.1−/− versus Nkx3.1+/+ prostate. (C) Diagram of the tissue recombination assay. Dissociated epithelium from seminal vesicle (SVE) or prostate (PE) is infected with a lentivirus expressing Nkx3.1 (or control). Mesenchyme from rat embryonic urogenital sinus is combined with the epithelium and grown under the renal capsule of host mice. (D and E) Representative tissue recombinants. (D) (Top) Whole-mount images. (Bottom) Hematoxylin and eosin (H&E) images. (E) Confocal images of immunofluorescence using the indicated antibodies. Scale bars represent 50 μm in (D) and 20 μm in (E). A summary of tissue recombinant data is provided in table S4.

Considering that loss of function of Nkx3.1 leads to aberrant prostate epithelial differentiation, we asked whether its gain of function in a nonprostatic epithelium is sufficient to induce prostate differentiation. Toward this end, we performed tissue recombination assays, in which relevant epithelial and mesenchymal tissues are recombined in vitro, followed by growth under the kidney capsule of host mice in vivo (Fig. 1C) (2). It is well established that prostate formation requires both epithelial and mesenchymal tissues, as well as an appropriate source of androgens (fig. S3), and that nonendodermal epithelium, even those that are androgen-regulated, such as seminal vesicle, do not form prostate in this assay (2).

To explore whether Nkx3.1 expression can induce prostate differentiation in tissue recombinants, we infected seminal vesicle epithelium with a lentivirus expressing Nkx3.1 (or the control vector) (Fig. 1C). As expected, tissue recombinants made with prostate epithelium (PE) generate prostate-like grafts, which are distinguished by their prostate-like ductal structures, histological appearance resembling prostate epithelium, and expression of markers of prostate differentiation, including Nkx3.1, probasin, FoxA1, and HoxB13 (N = 19 recombinants) (Fig. 1, D and E; fig. S4A; and table S4). Also as expected, tissue recombinants made with seminal vesicle epithelium (SVE) lacking Nkx3.1 generate seminal vesicle–like grafts, which are distinguished by their lack of discernible ductal morphology, histological appearance resembling seminal vesicle, and the absence of markers of prostate differentiation (N = 20 recombinants) (Fig. 1, D and E; fig. S4A; and table S4).

In contrast, tissue recombinants made with SVE expressing exogenous Nkx3.1 more closely resemble prostate than seminal vesicle. In particular, the Nkx3.1-expressing SVE grafts are distinguished by their appearance of prostate-like ductal structures, histological appearance resembling prostate, and expression of markers of prostate differentiation, including probasin, FoxA1, and HoxB13 (N = 26 recombinants) (Fig. 1, D and E; fig. S4A; and table S4); this was not the case for tissue recombinants made with SVE expressing an unrelated homeobox gene, Msx1 (N = 3 recombinants) (Fig. 1, D and E, and table S4). Moreover, expression profiling analyses showed that tissue recombinants made from SVE expressing Nkx3.1 were significantly enriched with a signature of prostate versus seminal vesicle (fig. S4, B and C). Nkx3.1 is thus sufficient to respecify a fully differentiated nonprostate epithelium to form prostate in vivo.

To study the underlying mechanisms, we established a cell-based assay using an immortalized human prostate cell line, RWPE1, which expresses low levels of NKX3.1, low levels of luminal cytokeratins, high levels of basal cell markers, and low levels of markers of prostate differentiation, such as androgen receptor (AR), FOXA1, PSA, TMPRSS2, and HOXB13 (Fig. 2, A to D). Infection of RWPE1 cells with a lentivirus expressing NKX3.1 resulted in high levels of NKX3.1 protein and robust DNA binding (Fig. 2, B and C). Expression profiling and Western blot analyses of the NKX3.1-expressing versus control RWPE1 cells revealed up-regulation of luminal cell markers (cytokeratins 8 and 18), down-regulation of basal cell markers (cytokeratin 5 and p63), and up-regulation of prostate differentiation markers (AR, FOXA1, PSA, TMPRSS2, and HOXB13) (Fig. 2, B and D). Furthermore, when combined with embryonic mesenchyme and grown under the renal capsule, NKX3.1-expressing RWPE1 cells generate prostate-like grafts that morphologically and histologically resemble prostate, including the presence of basal and luminal cell layers and expression of markers of prostate differentiation (N = 15 recombinants). In contrast, the control RWPE1 cells failed to grow in this assay (N = 12 recombinants) (Fig. 2E, fig. S5, and table S4). NKX3.1(T164A), which has a mutation in the homeodomain that impairs its DNA binding capacity (Fig. 2C) (9), did not induce prostate differentiation in vitro, nor did it promote prostate growth in tissue recombinants in vivo (N = 7 recombinants) (Fig. 2, B to E; fig. S5; and table S4). Therefore, the ability of NKX3.1 to induce prostate differentiation requires a functional DNA binding domain.

Fig. 2 Induction of prostate differentiation by NKX3.1 requires the homeodomain.

(A) Diagram of the experimental design. Human RWPE1 prostate epithelial cells are infected with a lentivirus expressing human NKX3.1, NKX3.1(T164A), or a control, followed by analyses in vitro (B to D) or recombined with mesenchyme and grown under the renal capsule of host mice (E). (B) Western blot analyses. Actin is a control for protein loading. (C) Gel retardation analysis done using nuclear extracts from RWPE1 cells expressing the control vector, NKX3.1, or NKX3.1 (T164A). The arrow indicates the free DNA probe. The experiments in (B) and (C) were each performed with three independent biological replicates; representative data are shown. (D) Heat map representations of selected differentially expressed genes; a complete list is provided in database S4. (E) Tissue recombinants showing whole-mount images, H&E, and immunofluorescence staining. The ruler shows cm scale; scale bars represent 50 μm in the H&E images and 20 μm in the immunofluorescence images. A summary of all tissue recombinants is provided in table S4.

Many of the functions of homeoproteins are mediated by protein-protein interactions that often occur through the homeodomain (10). Among NKX3.1-interacting proteins identified by mass spectrometry (Fig. 3A and fig. S6) (8) was G9a [also called EHMT2 (euchromatic histone lysine N-methyltransferase 2)], a histone methyltransferase that forms a complex with a related histone methyltransferase, GLP [also called EHMT1 (euchromatic histone lysine N-methyltransferase 1)], to promote dimethylation of lysine 9 on histone 3 (H3K9me2) (11). G9a is essential for embryonic development (12) and interacts with other homeoproteins to mediate differentiation (13, 14). In coimmunoprecipitation assays, NKX3.1 interacted with endogenous G9a, as well as GLP, which requires the homeodomain and is directly correlated with DNA binding by NKX3.1 (Fig. 3B and fig. S7, A to C). In contrast, NKX3.1 did not interact with other histone methyltransferases, including SUV39H1 (suppressor of variegation 3-9 homolog 1), which promotes trimethylation of lysine 9 on histone 3 (H3K9me3), and EZH2 (enhancer of zeste 2), which promotes trimethylation of lysine 27 on histone 3 (H3K27me3) (fig. S7, A and B).

Fig. 3 Induction of prostate differentiation by NKX3.1 is mediated through its interaction with the G9a histone methyltransferase.

(A and B) Nuclear extracts from RWPE1 cells expressing Flag-HA-NKX3.1 or the control were subjected to immunoprecipitation followed by mass spectrometry (A) or Western blot analysis (B) (see fig. S6). (A) Silver stain showing G9a interaction. Markers, as indicated. NS, nonspecific bands. (B) Immunoprecipitation followed by Western blot analysis. Input shows 5% of the total protein, and immunoprecipitation (IP) shows proteins recovered after IP using an antibody to Flag. Experiments were performed with three independent biological replicates; representative data are shown. (C) Diagram of experimental design for (D) and (E). Human RWPE1 prostate epithelial cells were infected with an NKX3.1-expressing lentivirus (expressing red fluorescent protein), followed by infection with an shRNA-expressing lentivirus (expressing green fluorescent protein). Coinfected cells were sorted by fluorescence-activated cell sorting, followed by analyses in vitro (D) or generation of tissue recombinants in vivo. (D) Western blot analysis. Experiments were performed with three independent biological replicates; representative data are shown. (E) Representative tissue recombinants showing whole-mount, H&E, and confocal images of immunofluorescence staining. Indicated is the kidney and the collagen plug (for the recombinants that did not grow) or the tissue recombinant. The ruler shows cm scale; scale bars represent 50μm in the H&E images and 20 μm in the immunofluorescence images. A summary of tissue recombinants is provided in table S4.

To study the functional relevance of the interaction between NKX3.1 and G9a, we coinfected RWPE1 cells with an NKX3.1-expressing lentivirus together with a short hairpin RNA (shRNA) to deplete G9a (shG9a) or SUV39H1 (shSUV39H1) as a control (Fig. 3, C to E, and fig. S5). These shRNAs reduced expression of G9a or SUV39H1, as well as their respective histone marks, H3K9me2 and H2K9me3, while not affecting expression of NKX3.1 (Fig. 3D). However, depletion of G9a, but not SUV39H1, impaired the ability of NKX3.1 to induce prostate differentiation, as evidenced by Western blot analysis of cultured cells, as well as prostate growth in the tissue recombinant assay in vivo (N = 9 recombinants) (Fig. 3, D and E; fig. S5; and table S4). These findings demonstrate that the interaction of NKX3.1 with G9a is required for induction of prostate differentiation.

Considering that induction of differentiation by NKX3.1 requires its homeodomain and corresponding DNA binding activity (fig. S7), we reasoned that these functions are likely to be mediated by gene transcription. Among NKX3.1 target genes that have predicted functions for differentiation and are conserved between mice and humans (fig. S8) (8), we focused on UTY, using EDEM2 as a control. In particular, G9a binds to the promoter of UTY, but not EDEM2, which is dependent on NKX3.1 binding and is required for NKX3.1-mediated up-regulation of UTY expression (Fig. 4A and fig. S9). Notably, UTY (KDM6c, ubiquitously transcribed tetratricopeptide repeat containing, Y-linked) is the male-specific paralog of UTX (KDM6a), a histone demethylase that is essential for viability and is frequently deregulated in cancer (1517). Although its functions as a histone demethylase are uncertain (16), UTY is essential for male fertility as well as the development and differentiation of male-specific organs (16), and it has been linked to prostate cancer (18).

Fig. 4 Induction of prostate differentiation by NKX3.1 is mediated by UTY, a male-specific paralog of histone demethylase UTX (KDM6c).

(A) Real-time polymerase chain reaction (PCR) showing expression of NKX3.1 target genes, UTY and EDEM2 (left). Chromatin immunoprecipitation quantitative PCR analysis of NKX3.1 binding (center) and G9a binding (right) to NKX3.1 target genes were performed. Analyses were performed with three independent biological replicates. Statistical analysis was done using a two-tailed t test; data are indicated as mean ± SD. (B) Diagram of the experimental design for (C) to (E). Human RWPE1 cells (C and D) or mouse tissues (E) were infected with an NKX3.1-expressing lentivirus, followed by infection with an shRNA (or controls). Cells were analyzed in vitro [human (C)] or in tissue recombinants [human (D) and mouse (E)]. (D) Western blot analysis of RPWE1 cells. [(D) and (E)] Representative tissue recombinants of human (D) and mouse (E) showing whole-mount, H&E, and confocal images of immunofluorescence staining. The ruler shows cm scale; scale bars represent 50 μm in the H&E images and 20 μm in the immunofluorescence images. A summary of tissue recombinants is provided in table S4.

To study the consequences of UTY depletion on prostate differentiation, we coinfected NKX3.1-expressing or control RWPE1 cells with an shRNA to UTY (shUTY) or EDEM2 (shEDEM2) as a control (Fig. 4, B to D, and fig. S5). Expression of these shRNAs reduced expression of UTY or EDEM2, respectively, while not affecting expression of NKX3.1 (Fig. 4C). Moreover, depletion of UTY, but not EDEM2, impaired the ability of NKX3.1 to induce prostate differentiation in vitro as well as in tissue recombinants in vivo (N = 8 recombinants) (Fig. 4, C and D, and table S4).

We next investigated whether Uty is also required for prostate specification in vivo by performing analogous experiments using the mouse tissue recombinant model. Depletion of Uty (shUty) in Nkx3.1-expressing SVE abrogated the ability of Nkx3.1 to generate prostate, as was evident by the resulting tissue recombinants (SVE + Nkx3.1 + shUty), which more closely resemble seminal vesicle than prostate (N = 8 recombinants) (Fig. 4E). Moreover, expression profiling of tissue recombinants generated from the Uty-depleted Nkx3.1-SVE revealed their significant enrichment in a gene signature comparing seminal vesicle versus prostate (fig. S10).

Cumulatively, our findings support a model in which NKX3.1 regulates the expression of a gene program associated with prostate differentiation, while simultaneously inhibiting the inappropriate expression of nonprostatic genes (fig. S11A). In particular, Nkx3.1 can respecify a fully differentiated mouse tissue to form prostate in vivo, and its expression in basal-like human prostate epithelial cells can promote their differentiation to luminal-like cells that form prostate in vivo. These functions of NKX3.1 are mediated by the coordinate actions of G9a and UTY (fig. S11, A and B). Notably, we find that G9a functions as a coregulator of NKX3.1, which is associated with activation, as well as repression, of NKX3.1 target genes. This further underscores the complexity of G9a function in transcriptional control. Indeed, although G9a is a histone methyltransferase for a “repressive” mark, it is active on euchromatin (11), and it has been shown that G9a can repress or activate transcription depending on the context (19, 20). This includes the glucocorticoid receptor (21, 22), which also interacts with NKX3.1, as assessed by mass spectrometry (fig. S6), and has been implicated in drug resistance in prostate cancer (23). Thus, we envision that our findings presage a role for G9a in both prostate differentiation and cancer.

Our findings also shed new light on UTY as an essential downstream mediator of NKX3.1 in prostate differentiation. Given the importance of UTY for male fertility, as well as for differentiation of male-specific organs (16, 24), our current study provides an example of how, in addition to the well-known role of androgen signaling, the promotion of “maleness” may be essential for prostate differentiation.

Last, there are still relatively few examples in which a single gene can respecify an otherwise fully differentiated epithelium to a new fate, as we have observed for NKX3.1. Notably, our findings regarding NKX3.1 in prostate are strikingly concordant with the functions of NKX2.1 in lung differentiation and lung cancer (2528). In particular, loss of function of NKX2.1 leads to impaired lung differentiation, which is associated with the derepression of an aberrant gene expression program (28), while, conversely NKX2.1 expression is associated with inhibition of lung metastases (26, 27). Consistent with the action of NKX3.1 in prostate, these actions of NKX2.1 in lung are dependent upon its level of expression. Thus, these observations suggest that NK-class homeobox genes function as key regulators of tissue-specific differentiation, as well as key gatekeepers whose diminution of expression in specific tissue contexts may enhance susceptibility to cancer.

Supplementary Materials

www.sciencemag.org/content/352/6293/1576/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S11

Tables S1 to S6

Databases S1 to S4

References (2944)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
Acknowledgments: We are grateful to A. Aytes, F. Constantini, E. Gelmann, D. Reinberg, and M. Shen for comments on the manuscript. We thank C. Bieberich (University of Maryland) for Nkx3.1 antisera. We acknowledge support from the JP Sulzberger Columbia Genome Center and the Proteomics Shared Resource, which are shared resources of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported in part by NIH/NCI grant P30 CA013696. This research is supported by funding to C.A.-S. from the National Cancer Institute (CA154293). A.D. was supported in part by the National Center for Advancing Translational Sciences, National Institutes of Health, grant UL1 TR000040. C.L.M. was supported by the Swiss National Science Foundation (PBBSP3_146959 and P300P3_151158). A.M. is a recipient of a Prostate Cancer Foundation Young Investigator Award. C.A.-S. is an American Cancer Society Research Professor supported in part by a generous gift from the F.M. Kirby Foundation.
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