We have performed a high throughput, in vivo genetic screen to identify kinases that permit androgen-dependent transformed prostate epithelial cells LHSR-AR cells to form tumors in female animals. In addition to known prostate cancer oncogenes and mediators of androgen independence mutated KRAS, constitutively active MEK, RAF1, ERBB2, AKT1, PIM1 and PIM2, overexpression of the Never In Mitosis A NIMA related kinase 6 NEK6 reproducibly yielded androgen-independent tumors. NEK6 is overexpressed in prostate cancer cell lines compared to their normal counterparts and is overexpressed in a subset of human prostate cancers. Expression of NEK6confers castration resistance to established tumors in male mice, and suppressing NEK6 expression restores sensitivity to castration. Castration-resistant tumors generated through NEK6 overexpression are predominantly squamous in histology and AR negative. Phosphoproteome analysis reveals the transcription factor FOXJ2 to be a novel substrate. The gene expression profile mediated by NEK6overexpression in tumors from castrated mice demonstrates elements of both differentiation and immune signaling, and a phosphomimetic form of FOXJ2 leads to transcriptions of newly identified NEK6 transcriptional targets. These studies reveal a novel mechanism of resistance in androgen pathway independent prostate cancer APIPC. Analysis of a genome-wide ORF screen for genes conferring androgen-independent proliferation of LNCaP cells in vitro suggests CREB5 as a novel mediator of castration resistance.