DNA methylation paradigm shift: 15-Lipoxygenase-1 upregulation in prostatic intraepithelial neoplasia and prostate cancer by atypical promoter hypermethylation

https://doi.org/10.1016/j.prostaglandins.2006.05.015Get rights and content

Abstract

Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4 + 3) and (3 + 4)], >7 with metastasis, (8–10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.

Introduction

Although the estimated US cases of diagnosed prostate cancer (PCa) in 2006 has slightly increased, as compared with previous years, a reduction in the number of deaths is projected. Nevertheless, it remains as one of the leading causes of cancer deaths among men in the United States [American Cancer Society, Facts and Figures 2006] in part because neither early onset intervention strategies for improving quality of life nor curative therapy for advanced disease exists [1]. The “baby boomer” generation will significantly exacerbate this major health problem. The age specific incidence of PCa increases after age 60 and in 2006–2007, 80 million “baby boomers” in the USA will approach this milestone. Other projections estimate that the numbers of new cases of PCa during the next 20 years will more than double and the number of men dying of the disease could double or triple [2]. These estimates underscore the urgent need for new biomarkers and therapeutic strategies that target the early stages of the disease.

Alterations in epigenetic activity can have a direct and profound effect on carcinogenesis. For example, aberrant methylation of CpG islands in the promoter region of tumor suppressor genes can lead to their transcriptional silencing and concomitant loss of tumor suppressor activity [3], [4]. There is also evidence that epigenetic alterations can also lead to up regulation of transcription [5]. Considering that epigenetic aberrations rather than genetic mutations play a key role in early PCa development, the reversibility of these changes with histone deacetylase and methylation inhibitors represents an attractive therapeutic option [6], [7], [8]. DNA methylation is an epigenetic alteration carried out by DNA methyltransferases (DNMT). These enzymes catalyze the covalent addition of a methyl group from a donor S-adenosyl-methionine to the 5 position of cytosine, predominantly within CpG dinucleotides. A major gap in understanding methylation dysregulation and neoplastic transformation is the lack of knowledge about the mechanisms underlying sequential changes in methylation patterns during the preneoplastic period in vivo. Current knowledge is based primarily on comparisons of methylation profiles between normal cells and tumor cells and it is not clear whether methylation instability in tumor cells stems arises from an isolated determining event, or from progressive alterations in heritable methylation patterns.

Studies from our laboratory [9], [10], [11], [12], [13] as well as from others [14], [15], [16], [17] have reported a key role for 15-LO-1 in several diseases including high grade prostatic intraepithelial neoplasia (HGPIN) and prostate tumors. Fifteen-LO-1 is a highly regulated, tissue- and cell-type-specific polyunsaturated fatty acid-peroxidating enzyme that has several functions, ranging from physiological membrane remodeling to the pathogenesis of atherosclerosis, inflammation and carcinogenesis. Expression of the 15-LO-1 protein is upregulated in PCa, which in combination with a diet high in omega-6 polyunsaturated fatty acids (PUFAs), leads to the formation of tumorigenic metabolite 13-hydroxyoctodecadenoic acid (13-S-HODE) [18], [19]. Due to the probable impact of 15-LO-1 on PCa development, targeting overexpression of 15-LO-1 expression may delay disease progression. However, what causes aberrant expression of 15-LO-1 in cancer cells remains unknown.

Human 15-LO-1 is located on chromosome 17p13.3 [13], a region which is frequently methylated in PCa [20]. Based on previously published studies on 5-LO and 15-LO-1 promoter methylation patterns and expression [21], [22], [23], we hypothesized that 15-LO-1 may also undergo methylation of its promoter region in PCa. Therefore, in this study, we began by examining the methylation and expression status of 15-LO-1 in normal, benign prostatic hyperplasic (BPH) cells and malignant PCa cell lines. We then examined the effect of DNA methyltransferase and histone deacetylase inhibitors on 15-LO-1 expression. Furthermore, we characterized the methylation profile of a specific CpG residue in the 15-LO-1 promoter, that when methylated correlated with 15-LO-1 expression in the cell lines, in cancer-associated HGPIN, PCa specimens, normal tissue adjacent to tumor and normal prostate tissue from cancer-free organ donors. Our findings demonstrated that the hypermethylation of an HpyCH4IV site in the 5′-flanking region −217 to −474 nt of the 15-LO-1 gene is associated with the transcriptional activation of the 15-LO-1 gene in PCa.

Section snippets

Cell lines and tissues

All cell lines were grown in a 5% CO2 incubator at 37 °C and 85% humidity. Primary prostate epithelial cells (PrEC) were maintained in PrEGM prostate epithelial cell medium (Clonetics, San Diego, CA). RWPE1, an immortalized normal prostate epithelial cell line and the PCa cell lines, LNCaP, PC-3, DU145 and MDAPCa2b were obtained from the American type culture collection (ATCC; Manassas, VA) and were maintained in the recommended medium. Los Angeles Prostate Cancer-4 (LAPC-4) PCa cells were

Relationship between 15-LO-1 mRNA expression and promoter methylation in human prostate cell lines

To examine the methylation and expression status of 15-LO-1, we used prostate cell lines representative of normal tissue as well as benign and malignant prostate disease. Based on real-time polymerase chain reaction (RT-PCR) analysis, 15-LO-1 mRNA was undetectable in the normal prostate cell lines PrEC (Fig. 1, lane 7) and RWPE1 (Fig. 1, lane 8). However, low levels were detected in BPH1, DU145, LAPC4 (Fig. 1, lanes 1–3) and PC3 (Fig. 1, lane 6) cells while high levels of 15-LO-1 mRNA were

Discussion

Epigenetic DNA methylation profoundly impacts carcinogenesis; thus, to fully comprehend the mechanisms underlying the etiology and progression of cancer will require a thorough exploration of the relationship between epigenetics and cancer. In addition, a more complete understanding of epigenetics has the potential to lead to the development of novel biomarkers that may significantly improve clinical cancer screening, risk assessment and treatment.

Epigenetic processes, such as methylation, have

Acknowledgements

We sincerely thank Dr. Moira Hitchens for critical reading and editing the manuscript. This work was fully supported by the Hillman Foundation Award to UPK and by the U.S. Army Department of Defense grant (W81XWH) and Edwin Beer program of the New York Academy of Medicine to DSOK. This work in no way reflects the opinion of the U.S. government.

References (45)

  • T.A.D.F. Bismar et al.

    Defining aggressive prostate cancer using a 12-gene model

    Neoplasia

    (2006)
  • J.M. Chan et al.

    The relative impact and future burden of prostate cancer in the United States

    J Urol

    (2004)
  • J.A. Hanson et al.

    Gene promoter methylation in prostate tumor-associated stromal cells

    J Natl Cancer Inst

    (2006)
  • J.E. De Larco et al.

    Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells

    PNAS

    (2003)
  • V.E. Steele et al.

    Potential use of lipoxygenase inhibitors for cancer chemoprevention

    Expert Opin Investig Drugs

    (2000)
  • N. Stern et al.

    Selective inhibition of angiotensin-II-mediated aldosterone secretion by 5-hydroxyeicosatetraenoic acid

    Endocrinology

    (1989)
  • U. Kelavkar et al.

    Concordant induction of 15-lipoxygenase-1 and mutant p53 expression in human prostate adenocarcinoma: correlation with Gleason staging

    Carcinogenesis

    (2000)
  • U. Kelavkar et al.

    The effect of 15-lipoxygenase-1 expression on cancer cells

    Curr Urol Rep

    (2002)
  • U.P. Kelavkar et al.

    Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis

    Carcinogenesis

    (2001)
  • H. Kamitani et al.

    Expression of 15-lipoxygenase-1 is regulated by histone acetylation in human colorectal carcinoma

    Carcinogenesis

    (2001)
  • U.P. Kelavkar et al.

    Effects of mutant p53 expression on human 15-lipoxygenase-promoter activity and murine 12/15-lipoxygenase gene expression: evidence that 15-lipoxygenase is a mutator gene

    Proc Natl Acad Sci USA

    (1999)
  • D.E. Blask et al.

    Melatonin-depleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats

    Cancer Res

    (2005)
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