Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMMs), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. While a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre|PtenFl/Fl) failed to progress beyond PIN and did not harbor genomic copy number alterations. By contrast, mice with both alterations (Hoxb13-MYC|Hoxb13-Cre|PtenFl/Fl or BMPC) developed lethal adenocarcinoma with distant metastases and widespread genome copy number alterations that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histological and genomic levels.

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate[1]. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.

The NKX3.1 gene located at 8p21.2 encodes a homeodomaincontaining transcription factor that acts as a haploinsufficient tumor suppressor in prostate cancer. Diminished protein expression of NKX3.1 has been observed in prostate cancer precursors and carcinomas. TOPORS is a ubiquitously expressed E3 ubiquitin ligase that can ubiquitinate tumor suppressor p53. Here we report interaction between NKX3.1 and TOPORS. NKX3.1 can be ubiquitinated by TOPORS in vitro and in vivo, and overexpression of TOPORS leads to NKX3.1 proteasomal degradation in prostate cancer cells. Conversely, small interfering RNA-mediated knockdown of TOPORS leads to an increased steady-state level and prolonged half-life of NKX3.1. These data establish TOPORS as a negative regulator of NKX3.1 and implicate TOPORS in prostate cancer progression.Prostate cancer is the second leading cause of cancer deaths and the most frequently diagnosed malignancy in American men (1). Prostate carcinomas are considered to arise from cancer precursors including prostatic intraepithelial neoplasia (PIN) 2 and proliferative inflammatory atrophy (2). Molecular alterations, including hereditary and somatic gene mutations, gene deletions, gene amplification, chromosomal rearrangements, as well as epigenetic changes, have been implicated in prostate cancer initiation and progression (3).The NK class homeobox gene NKX3.1 has been studied extensively over the past decade for its roles in prostate development and carcinogenesis (4). The expression of the murine Nkx3.1 gene is androgen-dependent and is restricted largely to prostate epithelial cells in adults (5-7). Deletion of Nkx3.1 by gene targeting leads to prostate ductal morphological defects, as well as prostatic dysplasia and hyperplasia that resembles human PIN (8 -10). Interestingly, heterozygous Nkx3.1 mice also develop hyperplasia and PIN-like lesions (8). The human NKX3.1 gene maps to 8p21.2 within a region where loss of heterozygosity occurs in PIN and is common in prostate carcinomas; however, no mutations have been found in the coding region of the NKX3.1 allele remaining (11,12). In light of these observations, NKX3.1 has been proposed to function as a haploinsufficient tumor suppressor. In support of a dose-dependent growth regulatory function of NKX3.1, reduced but not complete loss of NKX3.1 protein expression is now well documented in most human prostate cancer samples in a manner inversely correlated with Gleason score (13) and also with disease progression (14). Diminished NKX3.1 expression is thought to be an early event in prostate carcinogenesis, and reduced NKX3.1 immunostaining was observed in most proliferative inflammatory atrophy and PIN lesions analyzed (13). The diminished NKX3.1 expression may be partly attributed to selective CpG methylation of the NKX3.1 promoter in some prostate cancer cases (15). Nevertheless, quantitative analyses of mRNA and protein levels of NKX3.1 in prostate carcinoma lesions revealed a lack of concordance between mRNA and protein levels (13). In particular, decre...

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The prostate requires androgens for development and homeostasis. Prostate cancer shares this dependence, however progression to androgen-independence is common after androgen deprivation. There is considerable interest in achieving therapeutic gene expression after androgen ablation using prostate-specific promoters. Paradoxically, known prostate-restricted cis-regulatory elements are androgen dependent. Hoxb13 expression is restricted in adults to the prostate and colon, and robust Hoxb13 expression persists after castration. To locate regulatory elements conferring this expression pattern, a lacZ reporter was inserted into the Hoxb13 locus on a mouse genomic bacterial artificial chromosome. In transgenic mice, this construct recapitulated the Hoxb13 expression pattern, including expression after castration. Reporter gene activity was maintained during carcinogenesis in a prostate cancer model. Hoxb13 cis-regulatory elements provide a powerful tool to achieve androgen-independent transgene expression in the prostate and distal colon-specific expression in the gastrointestinal tract. These data establish a framework for high-resolution analyses of factors regulating Hoxb13.