Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common form of paediatric cancer and epigenetic aberrations are determinants of leukaemogenesis. The aim of this study was to investigate the methylation degree of a distinct phospholipase A2 receptor 1 (PLA2R1) promoter region in paediatric ALL patients and to evaluate its relevance as new biomarker for monitoring treatment response and burden of residual disease. The impact of PLA2R1 re-expression on proliferative parameters was assessed in vitro in Jurkat cells with PLA2R1 naturally silenced by DNA methylation. Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of 44 paediatric ALL patients. PLA2R1 methylation was analysed using digital PCR and compared to 20 healthy controls. Transfected Jurkat cells were investigated using cell growth curve analysis and flow cytometry. PLA2R1 was found hypermethylated in BM and PB from pre-B and common ALL patients, and in patients with the disease relapse. PLA2R1 methylation decreased along with leukaemic blast cell reduction during ALL induction treatment. In vitro analysis revealed an anti-proliferative phenotype associated with PLA2R1 re-expression, suggesting a tumour-suppressive function of PLA2R1. Collected data indicates that PLA2R1 promoter methylation quantitation can be used as biomarker for ALL induction treatment control, risk stratification, and early detection of ALL relapse.

Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR).

BACKGROUND: The analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. RESULTS: By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. CONCLUSIONS: We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.

Diverse effects of phospholipase A2 receptor expression on LNCaP and PC-3 prostate cancer cell growth in vitro and in vivo.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1's function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor's pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.

Epigenetic Mutation of the Succinate Dehydrogenase C Promoter in a Patient With Two Paragangliomas.

CONTEXT: Mutational inactivation of the succinate dehydrogenase (SDH) complex is a well-described cause of tumor development in pheochromocytomas/paragangliomas (PPGLs) and gastrointestinal stromal tumors (GISTs). Epigenetic inactivation of the SDHC gene is a more recently discovered phenomenon, which so far has only been described in GISTs and PPGLs from patients with Carney triad syndrome. CASE DESCRIPTION: A 33-year-old patient presented with two abdominal paragangliomas (PGLs) and an adrenocortical adenoma. Both PGLs showed high succinate:fumarate ratios indicative of SDHx mutations; however, no mutations in any of the known PPGL susceptibility genes were found in leucocyte or tumor DNA. We identified methylation of the SDHC promoter region in both PGLs, which coincided with decreased SDHC expression at mRNA and protein levels and a hypermethylated epigenomic signature (CpG island methylator phenotype). Low-level SDHC promoter methylation was also observed in the adenoma but not in normal adrenal tissue or blood, suggesting postzygotic somatic mosaicism for SDHC promoter methylation in the patient. CONCLUSIONS: This report provides evidence that SDHC promoter methylation can cause PGLs due to SDHC inactivation, emphasizing the importance of considering epigenetic changes and functional readouts in the genetic evaluation of patients not only with GISTs and Carney triad but also with PPGL.

Epigenetic control of group V phospholipase A2 expression in human malignant cells.

Secreted phospholipases A2 (sPLA2) are suggested to play an important role in inflammation and tumorigenesis. Different mechanisms of epigenetic regulation are involved in the control of group IIA, III and X sPLA2s expression in cancer cells, but group V sPLA2 (GV-PLA2) in this respect has not been studied. Here, we demonstrate the role of epigenetic mechanisms in regulation of GV-PLA2 expression in different cell lines originating from leukaemia and solid cancers. In blood leukocytes from leukaemic patients, levels of GV-PLA2 transcripts were significantly lower in comparison to those from healthy individuals. Similarly, in DU-145 and PC-3 prostate and CAL-51 and MCF-7 mammary cancer cell lines, levels of GV-PLA2 transcripts were significantly lower in relation to those found in normal epithelial cells of prostate or mammary. By sequencing and methylation-specific high-resolution melting (MS-HRM) analyses of bisulphite-modified DNA, distinct CpG sites in the GV-PLA2 promoter region were identified that were differentially methylated in cancer cells in comparison to normal epithelial and endothelial cells. Spearman rank order analysis revealed a significant negative correlation between the methylation degree and the cellular expression of GV-PLA2 (r = -0.697; p = 0.01). The effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on GV-PLA2 transcription in the analysed cells confirmed the importance of DNA methylation and histone modification in the regulation of the GV-PLA2 gene expression in leukaemic, prostate and mammary cancer cell lines. The exposure of tumour cells to human recombinant GV-PLA2 resulted in a reduced colony forming activity of MCF-7, HepG2 and PC-3 cells, but not of DU-145 cells suggesting a cell-type-dependent effect of GV-PLA2 on cell growth. In conclusion, our results suggest that epigenetic mechanisms such as DNA methylation and histone modification play an important role in downregulation of GV-PLA2 expression in cancer cells.

Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

BACKGROUND: It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines. METHODS: Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system. RESULTS: Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, -181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, -154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression. CONCLUSIONS: Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, -154, and -302d, as well as hsa-miR-141, -181b, and -181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.

Silibinin down-regulates expression of secreted phospholipase A2 enzymes in cancer cells.

BACKGROUND: Silibinin, a naturally-occurring flavonoid produced by milk thistle, possesses antioxidant, anti-inflammatory and cancer-preventive activities. In the current study, we examined the effects of silibinin on the expression of secreted phospholipase A2 (sPLA2) enzymes, especially those of group IIA (hGIIA), which play a crucial role in inflammation and carcinogenesis. MATERIALS AND METHODS: The effects of silibinin on sPLA2 expressions in human HepG2 hepatoma and PC-3 prostate cancer cells were analyzed using quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay technique. RESULTS: Silibinin inhibited the expression of hGIIA in unstimulated and cytokine-primed HepG2 and PC-3 cells. The mRNA levels of sPLA2 of groups IB, III and V were also significantly decreased by silibinin. Analyses of transcription factor activation suggest that nuclear factor-κB, but not specificity protein 1 (SP1) is implicated in the silibinin-mediated down-regulation of hGIIA. CONCLUSION: Silibinin exhibits inhibitory effects on basal and cytokine-induced expression of sPLA2s in cancer cells and therefore, may have the potential to protect against up-regulation of hGIIA and other sPLA2 isoforms during inflammation and cancer.

Deregulated expression of urokinase and its inhibitor type 1 in prostate cancer cells: role of epigenetic mechanisms.

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2'-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2±1.7%) in comparison to adjacent non-tumor tissue (6.0±0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.