Global levels of H3K27me3 track with differentiation in vivo and are deregulated by MYC in prostate cancer.

Cancer cells and stem cells share a number of biological characteristics including abundant amounts of decondensed chromatin. However, the molecular correlates and the factors involved in altering chromatin structure in cancer cells are not well known. Here, we report that less differentiated stem-like cells in the basal compartment of human and mouse prostate contain lower levels of the polycomb heterochromatin marker H3K27me3 than more differentiated luminal cells. This link to differentiated normal cells is also found in a number of other human and rodent tissues characterized by hierarchical differentiation. In addition to MYC's traditional role as a gene-specific transcription factor, recent studies indicate that MYC also affects global chromatin structure where it is required to maintain "open" or active chromatin. We now demonstrate that in both MYC-driven prostate cancers in mice and human prostate cancers, global levels of H3K27me3 are reduced in prostatic intraepithelial neoplasia and invasive adenocarcinoma lesions. Moreover, decreased levels of H3K27me3 correlate with increased markers of disease aggressiveness (eg, Gleason score and pathological stage). In vitro, experimentally forced reductions in MYC levels result in increased global levels of H3K27me3. These findings suggest that increased levels of decondensed chromatin in both normal progenitor cells and cancer cells are associated with global loss of H3K27me3, which is linked to MYC overexpression.

Myc enforces overexpression of EZH2 in early prostatic neoplasia via transcriptional and post-transcriptional mechanisms.

EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.

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.

Gene expression changes are age-dependent and lobe-specific in the brown Norway rat model of prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related enlargement of the prostate, characterized by increased proliferation of stromal and epithelial cells. Despite its prevalence, the etiology of BPH is unknown. METHODS: The Brown Norway rat is a model for age-dependent, lobe-specific hyperplasia of the prostate. Histological analyses of the dorsal and lateral lobes from aged rats reveal focal areas characterized by increased numbers of luminal epithelial cells, whereas the ventral lobe is unaffected. This study examined differential gene expression by lobe and age in the Brown Norway rat prostate. The objective was to identify genes with different levels of expression in the prostate lobes from 4-month (young) and 24-month (old) animals, and to subsequently link changes in gene expression to mechanisms of prostate aging. RESULTS: The number of age-dependent differentially expressed genes was greatest in the dorsal compared to the ventral and lateral lobes. Minimal redundancy was observed among the differentially expressed genes in the three lobes. Age-related changes in the expression levels of 14 candidate genes in the dorsal, lateral and ventral lobes were confirmed by quantitative RT-PCR. Genes that exhibited age-related differences in their expression were associated with proliferation, oxidative stress, and prostate cancer progression, including topoisomerase II alpha (Topo2a), aurora kinase B (Aurkb), stathmin 1 (Stmn1), and glutathione S-transferase pi. Immunohistochemistry for Topo2a, Aurkb, and Stmn1 confirmed age-related changes in protein localization in the lateral lobe of young and aged prostates. CONCLUSION: These findings provide clues to the molecular events associated with aging in the prostate.

Loss of Nkx3.1 expression in the transgenic adenocarcinoma of mouse prostate model.

BACKGROUND: The transgenic adenocarcinoma of mouse prostate (TRAMP) model has been extensively characterized at the histological and molecular levels, and has been shown to mimic significant features of human prostate cancer. However, the status of Nkx3.1 expression in the TRAMP model has not been elucidated. METHODS: Immunohistochemical analyses were performed using dorsal, lateral, and ventral prostate (VP) lobes from ages 6 to 30 weeks. Quantitative RT-PCR analyses were performed to determine relative mRNA expression. RESULTS: Heterogeneous loss of Nkx3.1 was observed in hyperplastic lesions of the ventral, dorsal, and lateral lobes. At 6 weeks of age, the ventral lobe displayed profound loss of Nkx3.1. Diminished Nkx3.1 protein was observed in well- to moderately-differentiated cancer lesions of all lobes. Poorly differentiated (PD) tumors stained negatively for Nkx3.1. Quantitative RT-PCR analyses revealed the presence of Nkx3.1 mRNA in each lobe at all ages, albeit reduced to variable levels. CONCLUSIONS: These data suggest that disease progression in the TRAMP model may be driven by loss of function of Nkx3.1, in addition to p53 and Rb. Lobe-specific disease progression in the TRAMP model correlates with the reduction of Nkx3.1 protein. Regulation of Nkx3.1 expression during tumorigenesis appears to occur by post-transcriptional and post-translational mechanisms.

Decreased NKX3.1 protein expression in focal prostatic atrophy, prostatic intraepithelial neoplasia, and adenocarcinoma: association with gleason score and chromosome 8p deletion.

NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC) and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in five of seven cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease.