NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells.

As the second leading cause of cancer deaths and the most frequently diagnosed malignancy in men, prostate cancer presents significant challenges from the perspective of both clinical and molecular oncology (28). As with all malignancies, the characterization of genetic changes associated with disease progression is a high priority. Human chromosome 8p has long been suspected to harbor one or more tumor suppressor genes involved in the etiology of prostate cancer based on analyses of allelic loss (7). Loss of heterozygosity of 8p21 is observed in a high percentage of intraepithelial prostatic neoplasias and early carcinoma lesions, strongly implicating this region in the initial stages of prostate carcinogenesis (18, 19, 25, 54). A substantial body of evidence points to an NK class homeobox gene as a leading 8p21 candidate for a prostate growth-regulatory gene involved in tumor initiation (48). Identified in mice as a prostate-restricted and androgen-regulated gene (9, 26, 43, 47, 48), nkx3.1 and its human homolog, NKX3.1, have been extensively characterized at the genetic level for links to prostate cancer. In humans, NKX3.1 maps to the minimal region of overlap among 8p21 loss-of-heterozygosity cases that have been analyzed to date (51). The Knudson two-hit model predicts that if NKX3.1 behaves as a classical tumor suppressor gene, then in prostate cancer cases with 8p21 loss, the remaining allele should incur an inactivating mutation (32). Several studies have demonstrated that in such cases the remaining NKX3.1 allele is wild type and is transcribed, arguing against a canonical tumor suppressor function (41, 55). However, a tumor suppression role for NKX3.1 is easily reconciled with more recent interpretations of tumor suppressor gene function that highlight the need to consider haploinsufficiency (16, 44, 45). It is becoming increasingly clear that gene dosage effects among many tumor suppressors, including p53 (53) and perhaps even Rb (61), can play an important role in the development of malignancy. A current model proposes that a loss-of-function mutation in a tumor suppressor gene confers a growth advantage that results in an expansion of cells carrying that initiating mutation (44). This scenario increases a target cell population in which the next of an estimated total of four to eight genetic changes required to support the evolution of a malignant state can occur. The importance of NKX3.1 as a dose-dependent regulator of prostate epithelial cell growth is strongly supported by analyses of nkx3.1 knockout mice (3, 8, 46, 52). Homozygous nkx3.1 mutant mice develop prostate epithelial hyperplasia and dysplasia that progresses with age (8, 46, 52), and lesions with histologic features strongly resembling human prostatic intraepithelial neoplasia develop in homozygous mice between 1 and 2 years of age (30). Importantly, both hyperplasia and prostatic intraepithelial neoplasia-like lesions also occur in a significant proportion of nkx3.1 heterozygous mutants (30). In conjunction with loss of one allele of the tumor suppressor gene pten, loss of one allele of nkx3.1 leads to high-grade prostatic intraepithelial neoplasia capable of progressing to invasive adenocarcinoma that metastasizes to lymph nodes (1, 31). Concomitant loss of a p27kip1 allele exacerbates the phenotype observed in pten/nkx3.1 double heterozygotes (22). Using a castration-regeneration system with nkx3.1 knockout mice, a battery of genes that are coregulated by androgens in an nkx3.1 dosage-dependent manner have been defined (38). These genes exhibit a range of sensitivities to nkx3.1 dosage, with some being shut off upon loss of one allele, while others are less dramatically altered. This study also clearly implicates nkx3.1 in growth control by demonstrating that in heterozygous nkx3.1 knockout mice, the exit of prostate epithelial cells from the cell cycle is delayed, resulting in a sharp increase in cell number. Immunohistochemical analyses of prostatic intraepithelial neoplasia lesions and prostate tumors have demonstrated that diminution or complete loss of NKX3.1 expression is a common event (11, 31). Curiously, several studies have shown that NKX3.1 mRNA levels do not diminish in tumors and may in fact increase with disease progression (41, 57) although concordance between NKX3.1 mRNA and protein has been reported in one recent study (33). These paradoxical and conflicting observations underscore the need to investigate the regulation of NKX3.1 at multiple levels. NKX3.1 is known to be a phosphoprotein, and phosphorylation has been shown to alter its DNA binding affinity in vitro (23). We initiated an investigation of NKX3.1 regulation focusing on posttranslational regulation by phosphorylation. In the work reported here, we have demonstrated that CK2 can phosphorylate NKX3.1 in vitro, shown that Thr89 and Thr93 are CK2 phosphoacceptor sites, and determined that protein kinase CK2 regulates the half-life of NKX3.1 in prostate tumor cells. We have also determined that NKX3.1 is degraded primarily through a proteasomal pathway, suggesting that phosphorylation by CK2 protects NKX3.1 from degradation. Our studies further indicate that a specific isoform of the CK2α′ catalytic subunit of CK2 is responsible for phosphorylating NKX3.1, providing new insights into the regulation of CK2 activity. Together, these observations establish a strong link between NKX3.1 and CK2 and may implicate CK2 in prostate malignancy.