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MYC genes are deregulated in a plurality of human cancers. Through direct and indirect mechanisms, the MYC network regulates the expression of > 15% of the human genome, including both protein-coding and noncoding RNAs. This complexity has complicated efforts to define the principal pathways mediating MYC's oncogenic activity. MYC plays a central role in providing for the bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. The rate-limiting enzyme in polyamine biosynthesis, ODC, is a bona fide MYC target, as are other regulatory enzymes in this pathway. A wealth of data link enhanced polyamine biosynthesis to cancer progression, and polyamine depletion may limit the malignant transformation of preneoplastic lesions. Studies with transgenic cancer models also support the finding that the effect of MYC on tumor initiation and progression can be attenuated through the repression of polyamine production. High-risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote the expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO and/or SAM486, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in preclinical models. Here, we review the potential clinical application of these and additional polyamine depletion agents to neuroblastoma and other advanced cancers in which MYC is operative. (Clin Cancer Res 2009;15(19):5956–61)

Purpose: Polyamines are important regulators of cell growth and death. The polyamine modulated factor-1 (PMF-1) is involved in polyamine homeostasis. After identifying an enriched CpG island encompassing the PMF1 promoter, we aimed at evaluating the clinical relevance of PMF1 methylation in bladder cancer. Experimental Design: The epigenetic silencing of PMF1 by hypermethylation was tested in bladder cancer cells (n = 11) after azacytidine treatment. PMF1 methylation status was evaluated in 507 bladder tumors and 118 urinary specimens of bladder cancer patients and controls. PMF1 protein expression was analyzed by immunohistochemistry on tissue arrays containing bladder tumors for which PMF1 methylation was assessed (n = 218). Results: PMF1 hypermethylation was associated with gene expression loss, being restored in vitro by a demethylating agent. An initial set of 101 primary frozen bladder tumors served to identify PMF1 hypermethylation in 88.1% of the cases. An independent set of 406 paraffin-embedded tumors also revealed a high PMF1 methylation rate (77.6%). PMF1 methylation was significantly associated with increasing stage (P = 0.025). Immunohistochemical analyses revealed that PMF1 methylation was associated with cytoplasmic PMF1 expression loss (P = 0.032). PMF1 protein expression patterns were significantly associated with stage (P < 0.001), grade (P < 0.001), and poor overall survival using univariate (P < 0.001) and multivariate (P = 0.011) analyses. Moreover, PMF1 methylation in urinary specimens distinguished bladder cancer patients from controls (area under the curve = 0.800). Conclusion: PMF1 was identified to be epigenetically modified in bladder cancer. The association of PMF1 methylation with tumor progression and its diagnostic ability using urinary specimens support including PMF1 assessment for the clinical management of bladder cancer patients.

The fate of any given mRNA has remained difficult to experimentally follow and has left mechanisms of unexpected gene expression phenomena in the dark. Examples are differential stability of mRNA expression from different promoters, and inhibition of expression in trans from co-transfected plasmids encoding N1-spermidine/spermine, acetyltransferase and a second unrelated gene. Recent advances in single molecule fluorescence in situ hybridization (smRNA FISH) now bring such central aspects of cellular mRNA metabolism into view. Streamlining both the design and synthesis of probe sets of multiple tiled singly labeled 20-mer oligonucleotides with balanced GC content have made most mRNAs targetable. In contrast to traditional cell-disruptive RNA analyses - northern blotting, QPCR, and microarrays - that yield average and relative information on the content of the probed mRNA, smRNA FISH affords discrete and cell-specific copy numbers. That added spatial dimension can be applied to provide cell-to-cell variability and the actual range of mRNA numbers in a given cell within the field of focus, or to examine defined subsets of cells. The hypusine pathway genes (EIF5A1, EIF5A2, DHPS, DOHH, and SAT1) are essential with deep evolutionary conservation. The gene products are tightly linked to the growth of the cell with aberrant expression in cells that have lost proliferation control, and in several cancers. SAT1, encoding N1-spermidine/spermine, acetyl-transferase, in particular plays a pivotal function in regulating polyamine levels and in turn determining the cells’ proliferative state. Recently, vectors episomally expressing SAT1 were found to inhibit the expression from co-transfected plasmids. Inhibition only occurred with enzymatically active SAT1. To gain a better understanding of how the expression of the hypusine pathway genes may contribute to the transformed phenotype, the fate of these mRNAs and those of unrelated reporter genes was examined by smRNA FISH. Single cell expression data for the hypusine pathway genes is presented and compared with previous results from northern analyses. Data is also presented on the further use of the probe sets to examine changes in the numerical and spatial expression in cells after manipulation of the hypusine status. Lastly, data on the expression - mRNA and protein - in cells co-transfected with vectors for SAT1 and unrelated reporter genes is presented, distinguishing between post-transcriptional events that may cause the inhibition. In summary, we show smRNA FISH as a suitable tool to disseminate post-transcriptional events for genes relevant to polyamine homeostasis and regulated protein synthesis in normal and aberrant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4209. doi:1538-7445.AM2012-4209