The essential role of methylthioadenosine phosphorylase in prostate cancer.

Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on the connected pathways, which are forced to increase metabolite production to maintain pools. The methionine salvage pathway recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools providing a mechanism to help mitigate metabolic stress associated with high flux through these pathways. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP), which, although commonly deleted in many cancers, is protected in prostate cancer. We report near universal retention of MTAP expression in a panel of human prostate cancer cell lines as well as patient samples. Upon metabolic perturbation, prostate cancer cell lines upregulate MTAP and this correlates with recovery of SAM levels. Furthermore, in a mouse model of prostate cancer we find that both normal prostate and diseased prostate maintain higher SAM levels than other tissues, even under increased metabolic stress. Finally, we show that knockdown of MTAP, both genetically and pharmacologically, blocks androgen sensitive prostate cancer growth in vivo. Our findings strongly suggest that the methionine salvage pathway is a major player in homeostatic regulation of metabolite pools in prostate cancer due to their high level of flux through the polyamine biosynthetic pathway. Therefore, this pathway, and specifically the MTAP enzyme, is an attractive therapeutic target for prostate cancer.

Dietary folate deficiency blocks prostate cancer progression in the TRAMP model.

Dietary folate is essential in all tissues to maintain several metabolite pools and cellular proliferation. Prostate cells, due to specific metabolic characteristics, have increased folate demand to support proliferation and prevent genetic and epigenetic damage. Although several studies have found that dietary folate interventions can affect colon cancer biology in rodent models, its impact on prostate is unknown. The purpose of this study was to determine whether dietary folate manipulation, possibly being of primary importance for prostate epithelial cell metabolism, could significantly affect prostate cancer progression. Strikingly, mild dietary folate depletion arrested prostate cancer progression in 25 of 26 transgenic adenoma of the mouse prostate (TRAMP) mice, in which tumorigenesis is prostate-specific and characteristically aggressive. The significant effect on prostate cancer growth was characterized by size, grade, proliferation, and apoptosis analyses. Folate supplementation had a mild, nonsignificant, beneficial effect on grade. In addition, characterization of folate pools (correlated with serum), metabolite pools (polyamines and nucleotides), genetic and epigenetic damage, and expression of key biosynthetic enzymes in prostate tissue revealed interesting correlations with tumor progression. These findings indicate that prostate cancer is highly sensitive to folate manipulation and suggest that antifolates, paired with current therapeutic strategies, might significantly improve treatment of prostate cancer, the most commonly diagnosed cancer in American men.

Plasma pentosidine: a potential biomarker in the management of multiple sclerosis.

BACKGROUND: The chronic inflammation associated with multiple sclerosis (MS) may lead to the upregulation of pentosidine. OBJECTIVES: This cross-sectional study compares plasma pentosidine levels among healthy controls (HCs) and patients with MS at different disease stages. The study also determines pentosidine's usefulness as a biomarker of MS disease activity and/or severity via its correlation with a number of indicators of MS disease. METHODS: Pentosidine levels were analyzed in 98 MS patients and 43 HCs using reverse-phase high-pressure liquid chromatography with fluorescence detection. RESULTS: Plasma pentosidine levels were significantly higher in MS patients when compared with HCs (p = 0.02). Patients on disease-modifying therapies (DMTs) had lower plasma pentosidine levels when compared with DMT-naïve patients (p = 0.01). Pentosidine plasma levels correlated with indicators of MS disease severity, including Extended Disability Status Scale (p = 0.03), MS Severity Scale (p = 0.01), and MS Functional Composite (p = 0.03). No correlation between pentosidine levels and age, rate of clinical relapse, and disease duration was observed. CONCLUSIONS: Our results suggest that pentosidine could be a novel, inflammatory biomarker in MS clinical practice. Longitudinal studies are warranted to determine any causal relationship between changes in plasma pentosidine levels and MS disease pathology. These studies may pave the way for use of advanced glycation end product (AGE) inhibitors and AGE-breaking agents as new therapeutic modalities in MS.

Mild folate deficiency induces genetic and epigenetic instability and phenotype changes in prostate cancer cells.

BACKGROUND: Folate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction. RESULTS: We studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion. CONCLUSIONS: This study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 microM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate in vivo, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.

Derangement of a factor upstream of RARalpha triggers the repression of a pleiotropic epigenetic network.

BACKGROUND: Chromatin adapts and responds to extrinsic and intrinsic cues. We hypothesize that inheritable aberrant chromatin states in cancer and aging are caused by genetic/environmental factors. In previous studies we demonstrated that either genetic mutations, or loss, of retinoic acid receptor alpha (RARalpha), can impair the integration of the retinoic acid (RA) signal at the chromatin of RA-responsive genes downstream of RARalpha, and can lead to aberrant repressive chromatin states marked by epigenetic modifications. In this study we tested whether the mere interference with the availability of RA signal at RARalpha, in cells with an otherwise functional RARalpha, can also induce epigenetic repression at RA-responsive genes downstream of RARalpha. METHODOLOGY/PRINCIPAL FINDINGS: To hamper the availability of RA at RARalpha in untransformed human mammary epithelial cells, we targeted the cellular RA-binding protein 2 (CRABP2), which transports RA from the cytoplasm onto the nuclear RARs. Stable ectopic expression of a CRABP2 mutant unable to enter the nucleus, as well as stable knock down of endogenous CRABP2, led to the coordinated transcriptional repression of a few RA-responsive genes downstream of RARalpha. The chromatin at these genes acquired an exacerbated repressed state, or state "of no return". This aberrant state is unresponsive to RA, and therefore differs from the physiologically repressed, yet "poised" state, which is responsive to RA. Consistent with development of homozygosis for epigenetically repressed loci, a significant proportion of cells with a defective CRABP2-mediated RA transport developed heritable phenotypes indicative of loss of function. CONCLUSION/SIGNIFICANCE: Derangement/lack of a critical factor necessary for RARalpha function induces epigenetic repression of a RA-regulated gene network downstream of RARalpha, with major pleiotropic biological outcomes.

A novel role for mitochondria in regulating epigenetic modification in the nucleus.

Epigenetic modification in the nuclear genome plays a key role in human tumorigenesis. In this paper, we investigated whether changes in the mtDNA copy number frequently reported to vary in a number of human tumors induce methylation changes in the nucleus. We utilized the Restriction Landmark Genomic Scanning (RLGS) to identify genes that undergo changes in their methylation status in response to the depletion and repletion of mtDNA. Our study demonstrates that depletion of mtDNA results in significant changes in methylation pattern of a number of genes. Furthermore, our study suggests that methylation changes are reversed by the restoration of mtDNA in cells otherwise lacking the entire mitochondrial genome. These studies provide the first direct evidence that mitochondria regulate epigenetic modification in the nucleus that may contribute to tumorigenesis.

A repressive epigenetic domino effect confers susceptibility to breast epithelial cell transformation: implications for predicting breast cancer risk.

Retinoic acid (RA) is a master epigenetic regulator that plays a pivotal role in both breast morphogenesis and development. Here, we show for the first time that RA, via the RA receptor alpha (RARalpha), epigenetically regulates in a concerted fashion the transcription of two RA-responsive genes, the RA receptor beta2 (RARbeta2) and the cellular retinol-binding protein 1 (CRBP1). Specifically, an impaired RA signal through RARalpha in human breast epithelial cells triggers a repressive epigenetic domino effect, involving first RARbeta2 and second CRBP1. The phenotype acquired by breast epithelial cells clearly implies that the resistance to RA-mediated growth inhibition precedes the acquisition of morphological epithelial transformation, thus supporting the occurrence of sequential transcriptional silencing of first RARbeta2 and second CRBP1. The identification of this epigenetic network mechanistically linking RARbeta2 and CRBP1 transcription provides the basis for devising more accurate epigenetic tests for the prediction of breast cancer risk.

Impaired retinoic acid (RA) signal leads to RARbeta2 epigenetic silencing and RA resistance.

Resistance to the growth-inhibitory action of retinoic acid (RA), the bioactive derivative of vitamin A, is common in human tumors. One form of RA resistance has been associated with silencing and hypermethylation of the retinoic acid receptor beta2 gene (RARbeta2), an RA-regulated tumor suppressor gene. The presence of an epigenetically silent RARbeta2 correlates with lack of the RA receptor alpha (RARalpha). Normally, RARalpha regulates RARbeta2 transcription by mediating dynamic changes of RARbeta2 chromatin in the presence and absence of RA. Here we show that interfering with RA signal through RARalpha (which was achieved by use of a dominant-negative RARalpha, by downregulation of RARalpha by RNA interference, and by use of RARalpha antagonists) induces an exacerbation of the repressed chromatin status of RARbeta2 and leads to RARbeta2 transcriptional silencing. Further, we demonstrate that RARbeta2 silencing causes resistance to the growth-inhibitory effect of RA. Apparently, RARbeta2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate that restoration of RA signal at a silent RARbeta2 through RARalpha leads to RARbeta2 reactivation. This report provides proof of principle that RARbeta2 silencing and RA resistance are consequent to an impaired integration of RA signal at RARbeta2 chromatin.