The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

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.

PD-1 expression is associated with a poor prognosis and is correlated with PD-L1 and Foxp3 expression in patients with gastric cancer.

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.

Intraductal carcinoma of the prostate is a marker of aggressive disease. However, intraductal carcinoma exists on a morphologic continuum with high grade prostatic intraepithelial carcinoma (PIN) and distinguishing intraductal carcinoma from PIN is a common diagnostic dilemma with significant clinical implications. We evaluated whether immunostains for PTEN and ERG can sensitively identify intraductal carcinoma and accurately distinguish it from high grade PIN. A combined immunostain for PTEN, ERG, p63 and CK903 was developed and validated. Radical prostatectomy specimens with lesions meeting criteria for intraductal carcinoma (n=45), intraductal cribriform proliferations falling short of intraductal carcinoma (n=15), and PIN lesions (n=39) were retrospectively identified and assessed for PTEN and ERG. Cytoplasmic PTEN loss was identified in 84% (38/45) of the intraductal carcinoma and 100% (15/15) of intraductal cribriform proliferation cases. In contrast, cytoplasmic PTEN loss was never observed in PIN (0/39) (p<0.0001). Of the 53 cases of intraductal carcinoma or intraductal cribriform proliferation with cytoplasmic PTEN loss, it was homogeneously lost in 42 cases (79%). Weak, focal nuclear positivity for PTEN was retained in 31 of these 42 cases (74%). ERG expression was identified in 58% (26/45) of intraductal carcinoma and 67% (10/15) of intraductal cribriform proliferations compared to 13% (5/39) of PIN. Concordance between the PTEN/ERG status of the intraductal carcinoma lesions and the concurrent invasive carcinoma was high (>95% and p<0.0001 for each), and substantially less for PIN and the concurrent invasive tumor (83% for PTEN and 67% for ERG; p=NS for each). Cytoplasmic PTEN loss occurs in the majority of intraductal carcinoma and intraductal cribriform proliferation cases. Cytoplasmic PTEN loss was never observed in PIN (100% specificity). Our study identifies PTEN loss as a potentially useful marker to distinguish intraductal carcinoma from PIN and provides a plausible molecular explanation for why intraductal carcinoma is associated with poor prognosis.

Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia.

Cervical cancer is a female-specific disease with a high incidence and mortality. MicroRNAs (miRNAs) are implicated in posttranscriptional regulation of gene expression and in the pathogenic mechanisms of cancer, suggesting their importance in diagnosis and treatment. miRNAs may have roles in the pathogenesis of cervical cancer based on the increases or decreases in several specific miRNAs found in patients with this disease. The miRNAs implicated in cervical cancer are miR-21, miR-126, and miR-143, and clinical application of these miRNAs for diagnosis and treatment is under investigation. Methods for diagnosis of cervical cancer include analysis of changes in the levels of specific miRNAs in serum and determination of aberrant hypermethylation of miRNAs. Supplementation of miR-143 or inhibition of miR-21 activity in vivo may be therapeutic strategy for cervical cancer. Previous approaches to development of siRNA as a drug have provided information for establishment of therapy based on these approaches, and an anti-miR-21 inhibitor has been developed. miRNAs also have effects on drug resistance and may be useful in combination therapy with other drugs.

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