Combined MYC Activation and Pten Loss Are Sufficient to Create Genomic Instability and Lethal Metastatic Prostate Cancer.

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations (CNA) 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 (GEMM), 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. Although 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∣Pten(Fl/Fl)) failed to progress beyond prostatic intraepithelial neoplasia and did not harbor genomic CNAs. By contrast, mice with both alterations (Hoxb13-MYC∣Hoxb13-Cre∣Pten(Fl/Fl), hereafter, BMPC mice) developed lethal adenocarcinoma with distant metastases and widespread genome CNAs 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 histologic and genomic levels.

Proline-mediated proteasomal degradation of the prostate-specific tumor suppressor NKX3.1.

Reduced expression of the homeodomain transcription factor NKX3.1 is associated with prostate cancer initiation and progression. NKX3.1 turnover requires post-translational modifications including phosphorylation and ubiquitination. Here, we demonstrate the existence of a non-canonical mechanism for NKX3.1 turnover that does not require ubiquitination. Using a structure-function approach, we have determined that the conserved, C-terminal 21-amino acid domain of NKX3.1 (C21) is required for this novel ubiquitin-independent degradation mechanism. Addition of C21 decreased half-life of enhanced green fluorescence protein (EGFP) by 5-fold, demonstrating that C21 constitutes a portable degron. Point mutational analyses of C21 revealed that a conserved proline residue (Pro-221) is central to degron activity, and mutation to alanine (P221A) increased NKX3.1 half-life >2-fold. Proteasome inhibition and in vivo ubiquitination analyses indicated that degron activity is ubiquitin-independent. Evaluating degron activity in the context of a ubiquitination-resistant, lysine-null NKX3.1 mutant (NKX3.1(KO)) confirmed that P221A mutation conferred additional stability to NKX3.1. Treatment of prostate cancer cell lines with a C21-based peptide specifically increased the level of NKX3.1, suggesting that treatment with degron mimetics may be a viable approach for NKX3.1 restoration.

A Hoxb13-driven reverse tetracycline transactivator system for conditional gene expression in the prostate.

BACKGROUND: Genetically engineered mouse models play important roles in analyses of prostate development and pathobiology. While constitutive genetic gain- and loss-of-function models have contributed significantly to our understanding of molecular events driving these processes, the availability of a tightly regulated inducible expression system could extend the utility of transgenic approaches. Here, we describe the development of a Tet-regulatory system that employs Hoxb13 transcriptional control elements to direct reverse tetracycline transactivator (rtTA) expression in the prostate. METHODS: Using recombineering technology, the rtTA gene was placed under Hoxb13 cis-regulatory transcriptional control in the context of a 218-kb bacterial artificial chromosome. F(1) offspring carrying the Hoxb13-rtTA transgene were bred to a Tetracycline operator-Histone 2B-Green Fluorescent Protein (TetO-H2BGFP) responder line. Detailed reporter gene expression analyses, including doxycycline (Dox) induction and withdrawal kinetics, were performed in Hoxb13-rtTA|TetO-H2BGFP double transgenic adult mice and embryos. RESULTS: Dox-dependent GFP expression was observed exclusively in the prostate and distal colon epithelia of double transgenic mice. Reporter gene mRNA was detected in the prostate within 6 hr of Dox exposure, and was extinguished within 24 hr after Dox withdrawal. Furthermore, Dox-induced reporter gene expression persisted after castration. CONCLUSIONS: The Hoxb13-rtTA transgenic system provides a powerful tool for conditional Tet operator-driven transgene expression in the normal prostate and during disease progression. Used in conjunction with other prostate pathology models, these mice will enable precise, temporally controlled analyses of gene function and can provide opportunities for detailed analyses of molecular events underlying prostate diseases.

Loss of Nkx3.1 expression in bacterial prostatitis: a potential link between inflammation and neoplasia.

NKX3.1 is a homeodomain protein that functions as a dosage sensitive prostate-specific transcription factor. Diminished NKX3.1 expression is associated with prostate epithelial cell proliferation in vitro and with increasing Gleason grade in patient samples. Mouse Nkx3.1 also functions as a negative regulator of prostate cell growth in prostate cancer models. Identifying biological and environmental factors that modulate NKX3.1 accumulation is therefore central to efforts aimed at elucidating prostate growth control mechanisms. To determine the effect of inflammation on Nxk3.1 accumulation, bacterial prostatitis was induced by intraurethral inoculation of a uropathogenic E. coli strain in mice. Nkx3.1 expression was profoundly reduced in infected prostate lobes and correlated with increased expression of a proliferation marker. Androgen receptor levels were also reduced in concert with Nkx3.1, and a marked increase in the basal cell marker p63 was observed. Analyses of the inflammatory infiltrate revealed a classic acute inflammatory response that attained characteristics of a chronic state within fourteen days postinoculation. Comparison of the four prostate lobes revealed clear differences in the extent of inflammation. These data demonstrate that acute inflammation in response to a bacterial agent in the prostate is associated with a significant diminution in the level of a key regulator of prostate cell proliferation. These observations provide a plausible mechanism whereby prostate inflammation may establish a local environment conducive to epithelial cell growth.

MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. 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.

A FOXA1-binding enhancer regulates Hoxb13 expression in the prostate gland.

Hoxb13 is robustly transcribed in derivatives of posterior endoderm including the colon, rectum, and the prostate gland. Transcriptional activity in the prostate persists unabated under conditions of androgen deprivation and throughout the course of disease progression in a mouse prostate cancer model. To elucidate the molecular basis of prostate-restricted transcriptional activation of Hoxb13, a bacterial artificial chromosome (BAC)-based reporter gene deletion analysis was performed in transgenic mice. Two regions downstream of the Hoxb13 coding region were found to be required to support transcriptional activity in the prostate but were completely dispensable for expression in the colon and rectum. Bioinformatic analyses of one region identified a 37-bp element conserved in mammals. This element, which bears two potential binding sites for Forkhead class transcription factors, is occupied by FOXA1 in a human prostate cancer cell line. Precise replacement of this enhancer with an extended LoxP site in the context of a 218,555-bp BAC reporter nearly extinguished Hoxb13-mediated transcriptional activity in the mouse prostate. These data demonstrate that FOXA1 directly regulates HOXB13 in human prostate epithelial cells, and show that this prostate-specific regulatory mechanism is conserved in mice.

Hoxb13 regulatory elements mediate transgene expression during prostate organogenesis and carcinogenesis.

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.

Ubiquitination by TOPORS regulates the prostate tumor suppressor NKX3.1.

The NKX3.1 gene located at 8p21.2 encodes a homeodomain-containing 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.

Distinct regulatory elements mediate the dynamic expression pattern of Nkx3.1.

Loss of Nkx3.1 function in mice results in defects in prostate development and epithelial hyperplasia, indicating that this gene plays important roles in both the initiation and maintenance of prostate differentiation. In humans, decreased NKX3.1 expression is associated with the progression of prostate cancer. Despite these roles in prostate development and disease, the transcriptional regulation of Nkx3.1 has not been systematically addressed. A reporter gene approach in transgenic mice was used to identify regulatory regions that dictate the expression pattern of Nkx3.1. A 32-kb DNA fragment from the Nkx3.1 locus that specifies the expected expression pattern during embryogenesis and postnatal life has been identified. Deletion analyses demonstrated that cis-regulatory elements that mediate expression in distinct sites are separable. A 5-kb fragment downstream of the Nkx3.1 coding region contains elements that support expression in the prostate and bulbourethral glands, whereas an upstream fragment contains elements that direct expression in somites and testes. Reporter gene expression analyses also revealed several previously unknown sites of Nkx3.1 expression in males, including urethral glands, glandular cells in the urethral diverticulum and basal epithelial cells in the prostate. In addition, these analyses revealed Nkx3.1 expression in female urethral glands. The identification of Nkx3.1 cis-regulatory elements provides a unique starting point to dissect signaling pathways involved in prostate organogenesis and pathogenesis and provides a system to perturb gene expression throughout prostate development.