Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6

Abstract

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR.

Figure 1

NEK6 confers androgen-independent tumor formation in a xenograft model of androgen-dependent prostate cancer. A. Effects of NEK6 expression on the rate of tumor formation at 60 d in the indicated LHSR-AR cells in female and castrated mice. Of note, if the tumor denoted with a is excluded, the difference in rate of tumor formation between parental LHSR-AR cells and cells expressing NEK6 in castrated mice remains statistically significant (p=0.009). B. The effects of expressing NEK6 on the rate of tumor formation at 60 d for parental LHMK-AR cells and cells expressing NEK6 in female mice. C. Left: Inducible expression of NEK6 in vitro at 48 hrs after addition of doxycycline Right: Waterfall plot of change in tumor volume at 30 d after castration (compared to prior to castration) of xenograft tumors derived from parental LHSR-AR cells and cells with inducible NEK6 expression formed in male mice.

Figure 2

A. NEK6-mediated androgen-independent tumors are primarily squamous in histology and AR negative. Sections of tumors derived from parental LHSR-AR cells expressing GFP in male mice, and cells expressing NEK6 in female and castrated mice were stained with AR antibody (brown). B. Sections of tumors derived from LHSR-AR cells overexpressing NEK6 in male mice with implanted testosterone pellet harvested prior to castration (Day 0) and at 4, 8, and 12 d after castration. C. Immunohistochemical staining at 20× magnification for NEK6 in prostate cancer tissue microarrays. Low (left and middle) and high grade (right) cases are represented, each core showing tumor infiltrating in between benign glands with higher expression seen in the tumor.

Figure 3

NEK6 is overexpressed in several prostate cancer cell lines compared to immortalized (RWPE, LH) and transformed (LHSR-AR) prostate epithelial cells. A. Left: Expression of NEK6 and AR in prostate cell lines with Hsp90 as loading control; Right: NEK6 expression in LHSR-AR cells (lane 1), LHSR-AR cells overexpressing NEK6 (lane 2), and in VCaP and LNCaP cells. B. Expression of NEK6 in CL-1, PC-3, and DU145 cells with doxycycline inducible expression of 2 shRNAs targeting NEK6 or a control targeting lacZ in the presence or absence of doxycycline (CL-1 and PC-3) or with doxycycline in the presence or absence of growth factor stimulation (DU145). C. Proliferation curves of cells cultured in the presence of doxycycline with cells in 60 mm plates, split and replated every 2–3 d as indicated. The average of 3 replicates with error bars is shown.

Figure 4

A. Immunoblots of protein lysates of xenograft tumors derived from LHSR-AR cells transduced with doxycycline-inducible constructs (in the pTRIPz vector) for the expression of GFP or NEK6 in male mice, with doxycycline maintained in the diet at time of harvest (+dox) or with doxycycline diet removed 7 d prior to harvest (−dox). Tumors were harvested from non-castrated mice (day 0) or 2 or 5 d after castration. Short and long exposures of the NEK6 immunoblot are shown. B. Gene Set Enrichment Analysis performed on the gene expression signature mediated by NEK6 overexpression at d 5 after castration (GSEA pre-ranked based on ratio of classes to tumors without NEK6 overexpression). The top two curated gene sets (C2) from the molecular signatures database (MSigDB) correlated with the NEK6 signature are shown. C. Left: Genes downregulated in the control (−dox) tumors after castration (fold change <−1.5, signal-to-noise <−1 in the comparison of tumors harvested at d 5 and day 2) were plotted against the NEK6 signature at d 5 using GSEA pre-ranked. Right: Venn diagram of genes downregulated in control tumors after castration intersected with genes upregulated by NEK6 at day 5 (fold change >1.5, signal-to-noise >1 in the comparison of +dox to −dox tumors). The GO terms most highly enriched in this overlap are cytokine-mediated and type I interferon signaling pathways (p=2×10⁻⁵).

Figure 5

A. Summary of phosphopeptides (categorized by phosphorylation motif) found to be enriched in cells expressing wild-type NEK6 under a doxycycline-inducible promoter as compared to kinase-dead NEK6 or cells not treated with doxycycline. B. NEK6 can phosphorylate NCOA5 and FOXJ2 in vitro at the sites discovered in the phosphoproteomic screen. 293T cells were transfected with expression constructs for wild-type and mutant (S-to-D) versions of NCOA5 or FOXJ2 with a C-terminal V5 tag and immunoprecipitated with anti-V5 antibody. Eluates from 1/5 of the beads were assayed by V5 immunoblot; the remaining 4/5 was subjected to on-bead in vitro kinase assay with recombinant active GST-NEK6 (Sigma).

Figure 6

A+B. Mapping NEK6 phosphorylation sites on FOXJ2 and NCOA5. 293T cells were transfected with expression constructs for wild-type and mutant (S-to-D) versions of NCOA5 or FOXJ2 with a C-terminal V5 tag and immunoprecipitated with anti-V5 antibody; + indicates a mutation is present at that residue, − indicates that the residue is wild-type (lane 1 of each blot represents the wild-type protein). 5% of the input assayed by V5 immunoblot is shown in the left panels; the kinase assay is shown in the right panels. FOXJ2 has 10 S/T residues of the previously reported NEK6 motif L/F/W/Y-X-X-pS/pT-F/W/Y/M/L/I/V/R/K; the six residues in FOXJ2 (or homologous residues in FOXJ3) that had been previously demonstrated to be phosphorylated in phosphosite.org were assayed here. NCOA5 has 5 S/T residues in this NEK6 phosphorylation motif; however, mutating Ser201 to aspartic acid dramatically decreased exogenous NCOA5 expression, so the remaining four sites were tested in this assay. C. Kinase assay performed for wild-type and (D×3) forms of FOXJ2 and NCOA5 in the presence (+) or absence (−) of active recombinant GST-NEK6 in the assay.

Figure 7

A. RNA-Seq was performed from 3 tumors inducibly expressing phosphomimetic forms of FOXJ2 or NCOA5 with continued doxycycline expression, and 3 matched tumors seven days post doxycycline withdrawal, 5 days after castration. Shown are plots demonstrating correlation of genes upregulated by these phosphomimetic forms with the NEK6 signature at day 5 by GSEA pre-ranked. B. Venn diagram of genes upregulated by NEK6 with genes upregulated by FOXJ2(D×3) at day 5 after castration (fold change >1.5, signal-to-noise >1 in the comparison of +dox to −dox tumors). C. Immunoblot of protein lysates of LHSR-AR cells transduced with doxycycline-inducible constructs (in the pTRIPz vector) for the expression of phosphorylation-deficient (A×3), wild-type, and phosphomimetic (D×3) forms of FOXJ2 treated with or without doxycycline for 48 hours. * indicates a background band used as a loading control. D. Gene expression of FOXJ2, TPPP3 and PSCA by quantitative RT-PCR (as fold-change from control) in cells expressing (A×3), wild-type and (D×3) forms of FOXJ2 under a doxycycline-inducible promoter treated with or without doxycycline for 48 hours. The average of 3 technical replicates (with error bars representing the standard deviation) is shown.