Tracy Murray Stewart

Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes

Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.

Novel Oligoamine Analogues Inhibit Lysine-Specific Demethylase 1 and Induce Reexpression of Epigenetically Silenced Genes

Purpose: Abnormal DNA CpG island hypermethylation and transcriptionally repressive histone modifications are associated with the aberrant silencing of tumor suppressor genes. Lysine methylation is a dynamic, enzymatically controlled process. Lysine-specific demethylase 1 (LSD1) has recently been identified as a histone lysine demethylase. LSD1 specifically catalyzes demethylation of mono– and dimethyl–lysine 4 of histone 3 (H3K4), key positive chromatin marks associated with transcriptional activation. We hypothesized that a novel class of oligoamine analogues would effectively inhibit LSD1 and thus cause the reexpression of aberrantly silenced genes. Experimental Design: Human colorectal cancer cells were treated with the oligoamines and changes in mono- and dimethyl-H3K4 and other chromatin marks were monitored. In addition, treated cells were evaluated for the reexpression of the aberrantly silenced secreted frizzled-related proteins (SFRP) Wnt signaling pathway antagonist genes. Finally, the effects of the LSD1 inhibitors were evaluated in an in vivo xenograft model. Results: Treatment of HCT116 human colon adenocarcinoma cells in vitro resulted in increased H3K4 methylation and reexpression of silenced SFRP genes. This reexpression is also accompanied by a decrease in H3K9me2 repressive mark. Importantly, cotreatment with low doses of oligoamines and a DNA methyltransferase inhibitor highly induces the reexpression of the aberrantly silenced SFRP2 gene and results in significant inhibition of the growth of established tumors in a human colon tumor model in vivo. Conclusions: The use of LSD1-inhibiting oligoamine analogues in combination with DNA methyltransferase inhibitors represents a highly promising and novel approach for epigenetic therapy of cancer. (Clin Cancer Res 2009;15(23):7217–28)

Cloning and Characterization of Human Polyamine-modulated Factor-1, a Transcriptional Cofactor That Regulates the Transcription of the Spermidine/Spermine N 1-Acetyltransferase Gene

The increased transcription and ultimate superinduction of the spermidine/spermineN 1-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of ∼1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of ∼20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.

Induction of the PAOh1/SMO polyamine oxidase by polyamine analogues in human lung carcinoma cells

PurposeThe induction of polyamine catabolism has been directly associated with the cytotoxic response of various tumor types to the antitumor polyamine analogues. Initially, human polyamine catabolism was assumed to be under the control of a rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (PAO). We have recently cloned a new polyamine analogue-inducible human polyamine oxidase (PAOh1/SMO) that efficiently uses spermine as a substrate. The induction of PAOh1/SMO in response to multiple polyamine analogues was examined in representative lung tumor cell lines.MethodsRepresentatives of three different classes of antitumor polyamine analogues were examined for their ability to induce PAOh1/SMO.ResultsThe human adenocarcinoma line, NCI A549 was found to be the most responsive line with respect to induction of PAOh1/SMO in response to analogue exposure. Similar to previous observations with SSAT expression, PAOh1/SMO induction was found to occur primarily in non-small-cell lung cancers cell lines. Using a series of polyamine analogues, it was found that the most potent inducers of PAOh1/SMO possessed multiple three-carbon linkers between nitrogens, as typified by N1,N11-bis(ethyl)norspermine.ConclusionsSince PAOh1/SMO is an analogue-inducible enzyme that produces H2O2 as a metabolic product, it may play a significant role in determining the sensitivity of various human tumors to specific polyamine analogues.

The role of polyamine catabolism in anti-tumour drug response

Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.

Polyamine-modulated factor 1 binds to the human homologue of the 7a subunit of the Arabidopsis COP9 signalosome: Implications in gene expression

Polyamines have been identified to play a role in the transcription of various growth-related genes. The recently discovered polyamine responsive element and the associated trans-acting proteins involved in polyamine-regulated transcription have provided a model system for the study of the role of polyamines in transcription. Polyamine-modulated factor 1 (PMF-1) was identified as one of the transacting factors that binds to NF-E2 related factor-2 (Nrf-2) to regulate the transcription of spermidine/spermine N(1)-acetyltransferase (SSAT). The possibility that PMF-1 also binds to other proteins involved in transcriptional regulation cannot be ruled out. Using a yeast two-hybrid strategy, it was found that PMF-1 binds to a human homologue of the Arabidopsis COP9 signalosome subunit 7a (CSN 7) protein. In the present study, we describe human CSN 7, a 275-amino-acid- containing protein that may have a direct role in regulating gene expression. CSN 7 and PMF-1 bind to each other, as well as compete with each other for binding to Nrf-2. This competition for Nrf-2 binding and interaction with each other is implicated in the regulation of SSAT transcription. CSN 7 possesses a C-terminal coiled-coil domain similar to the domain that mediates the interaction between PMF-1 and Nrf-2, suggesting that coiled-coil domains also mediate the interaction between CSN 7 and PMF-1. Since CSN 7 does not contain a DNA-binding domain, its effects on transcription must occur in conjunction with binding to other proteins. The results presented here demonstrate that PMF-1 and Nrf-2 can act as protein partners of CSN 7.

Reduction in polyamine catabolism leads to spermine-mediated airway epithelial injury and induces asthma features

Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the underlying mechanism of epithelial injury remains poorly explored. Although high levels of polyamines, mainly spermine, have been found in asthma and comorbidity, their role in airway epithelial injury and the cause of their altered levels in asthma have not been explored.