The crucial role of epigenetic regulation in breast cancer anti-estrogen resistance: Current findings and future perspectives.

Breast cancer (BC) cell de-sensitization to Tamoxifen (TAM) or other selective estrogen receptor (ER) modulators (SERM) is a complex process associated with BC heterogeneity and the transformation of ER signalling. The most influential resistance-related mechanisms include modifications in ER expression and gene regulation patterns. During TAM/SERM treatment, epigenetic mechanisms can effectively silence ER expression and facilitate the development of endocrine resistance. ER status is efficiently regulated by specific epigenetic tools including hypermethylation of CpG islands within ER promoters, increased histone deacetylase activity in the ER promoter, and/or translational repression by miRNAs. Over-methylation of the ER α gene (ESR1) promoter by DNA methyltransferases was associated with poor prognosis and indicated the development of resistance. Moreover, BC progression and spreading were marked by transformed chromatin remodelling, post-translational histone modifications, and expression of specific miRNAs and/or long non-coding RNAs. Therefore, targeted inhibition of histone acetyltransferases (e.g. MYST3), deacetylases (e.g. HDAC1), and/or demethylases (e.g. lysine-specific demethylase LSD1) was shown to recover and increase BC sensitivity to anti-estrogens. Indicated as a powerful molecular instrument, the administration of epigenetic drugs can regain ER expression along with the activation of tumour suppressor genes, which can in turn prevent selection of resistant cells and cancer stem cell survival. This review examines recent advances in the epigenetic regulation of endocrine drug resistance and evaluates novel anti-resistance strategies. Underlying molecular mechanisms of epigenetic regulation will be discussed, emphasising the utilization of epigenetic enzymes and their inhibitors to re-program irresponsive BCs.

Blood-Based DNA Methylation Analysis by Multiplexed OBBPA-ddPCR to Verify Indications for Prostate Biopsies in Suspected Prostate Cancer Patients.

Current prostate carcinoma (PCa) biomarkers, including total prostate-specific antigen (tPSA), have unsatisfactory diagnostic sensitivity and specificity resulting in overdiagnosis and overtreatment. Previously, we described an optimised bias-based preamplification-digital droplet PCR (OBBPA-ddPCR) technique, which detects tumour DNA in blood-derived cell-free DNA (cfDNA) of cancer patients. The current study investigated the performance of newly developed OBBPA-ddPCR-based biomarkers. Blood plasma samples from healthy individuals (n = 90, controls) and PCa (n = 39) and benign prostatic hyperplasia patients (BPH, n = 40) were analysed. PCa and BPH patients had tPSA values within a diagnostic grey area of 2-15 ng/mL, for whom further diagnostic validation is most crucial. Methylation levels of biomarkers RASSF1A, MIR129-2, NRIP3, and SOX8 were found significantly increased in PCa patients compared to controls. By combining classical PCa risk factors (percentage of free PSA compared to tPSA (QfPSA) and patient's age) with cfDNA-based biomarkers, we developed PCa risk scores with improved sensitivity and specificity compared to established tPSA and QfPSA single-marker analyses. The diagnostic specificity was increased to 70% with 100% sensitivity for clinically significant PCa patients. Thus, prostate biopsies could be avoided for 28 out of 40 BPH patients. In conclusion, the newly developed risk scores may help to confirm the clinical decision and prevent unnecessary prostate biopsy.

Epigenetic drugs in somatostatin type 2 receptor radionuclide theranostics and radiation transcriptomics in mouse pheochromocytoma models.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are catecholamine-producing tumors. In inoperable and metastatic cases, somatostatin type 2 receptor (SSTR2) expression allows for peptide receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE. Insufficient receptor levels, however, limit treatment efficacy. This study evaluates whether the epigenetic drugs valproic acid (VPA) and 5-Aza-2'-deoxycytidine (DAC) modulate SSTR2 levels and sensitivity to [177Lu]Lu-DOTA-TATE in two mouse PCC models (MPC and MTT). Methods: Drug-effects on Sstr2/SSTR2 were investigated in terms of promoter methylation, mRNA and protein levels, and radiotracer binding. Radiotracer uptake was measured in subcutaneous allografts in mice using PET and SPECT imaging. Tumor growth and gene expression (RNAseq) were characterized after drug treatments. Results: DAC alone and in combination with VPA increased SSTR2 levels along with radiotracer uptake in vitro in MPC (high-SSTR2) and MTT cells (low-SSTR2). MTT but not MPC allografts responded to DAC and VPA combination with significantly elevated radiotracer uptake, although activity concentrations remained far below those in MPC tumors. In both models, combination of DAC, VPA and [177Lu]Lu-DOTA-TATE was associated with additive effects on tumor growth delay and specific transcriptional responses in gene sets involved in cancer and treatment resistance. Effects of epigenetic drugs were unrelated to CpG island methylation of the Sstr2 promoter. Conclusion: This study demonstrates that SSTR2 induction in mouse pheochromocytoma models has some therapeutic benefit that occurs via yet unknown mechanisms. Transcriptional changes in tumor allografts associated with epigenetic treatment and [177Lu]Lu-DOTA-TATE provide first insights into genetic responses of PCCs/PGLs, potentially useful for developing additional strategies to prevent tumor recurrence.

Treatment of Pheochromocytoma Cells with Recurrent Cycles of Hypoxia: A New Pseudohypoxic In Vitro Model.

Continuous activation of hypoxia pathways in pheochromocytomas and paragangliomas (PPGLs) is associated with higher disease aggressiveness, for which effective treatment strategies are still missing. Most of the commonly used in vitro models lack characteristics of these pseudohypoxic tumors, including elevated expression of hypoxia-inducible factor (HIF) 2α. To address this shortcoming, we investigated whether recurrent hypoxia cycles lead to continuous activation of hypoxia pathways under normoxic conditions and whether this pseudohypoxia is associated with increased cellular aggressiveness. Rat pheochromocytoma cells (PC12) were incubated under hypoxia for 24 h every 3-4 days, up to 20 hypoxia-reoxygenation cycles, resulting in PC12 Z20 cells. PC12 Z20 control cells were obtained by synchronous cultivation under normoxia. RNA sequencing revealed upregulation of HIF2α in PC12 Z20 cells and a pseudohypoxic gene signature that overlapped with the gene signature of pseudohypoxic PPGLs. PC12 Z20 cells showed a higher growth rate, and the migration and adhesion capacity were significantly increased compared with control cells. Changes in global methylation, together with the pseudohypoxic conditions, may be responsible for the increased aggressiveness of this new model. The established sub-cell line with characteristics of pseudohypoxic PPGLs represent a complementary model for further investigations, for example, with regard to new therapeutic approaches.

Increased Sensitivity of Detection of RASSF1A and GSTP1 DNA Fragments in Serum of Prostate Cancer Patients: Optimisation of Diagnostics Using OBBPA-ddPCR.

Identification of aberrant DNA methylation is a promising tool in prostate cancer (PCa) diagnosis and treatment. In this study, we evaluated a two-step method named optimised bias-based preamplification followed by digital PCR (OBBPA-dPCR). The method was used to identify promoter hypermethylation of 2 tumour suppressor genes RASSF1A and GSTP1 in the circulating cell-free DNA (cfDNA) from serum samples of PCa patients (n = 75), benign prostatic hyperplasia (BPH, n = 58), and healthy individuals (controls, n = 155). The PCa cohort was further subdivided into subgroups comprising (I) patients with Gleason Scores (GS) ≤ 7 (n = 55), (II) GS ≥ 8 (n = 10), and (III) patients with metastatic PCa diagnosis (n = 10). We found that RASSF1A methylation levels were significantly increased in all 3 PCa subgroups compared to the controls and BPH cohorts (p < 0.01 for all comparisons). Fractional abundances of methylated GSTP1 DNA fragments were significantly increased in subgroup III of metastatic PCa patients (p < 0.001). RASSF1A methylation analysis was found to be beneficial as a complementary biomarker where further diagnostic validation is most crucial. In combination with free PSA, RASSF1A methylation status helps to identify PCa patients with GS ≥ 8 and grey-zone total PSA values between 2-10 ng/mL. In our study, PCR biases between 80-90% were sufficient to detect minute amounts of tumour DNA with high signal-to-noise ratios as well as high analytical sensitivity and specificity. Both RASSF1A and GSTP1 exhibited strongly increased DNA methylation levels in all metastatic PCa patients. Our data indicates a superior sensitivity of epigenetic biomarker analyses in early detection of PCa metastases that should also help to improve PCa therapy.

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.

Sphingosine kinase and sphingosine-1-phosphate receptor signaling pathway in inflammatory gastrointestinal disease and cancers: A novel therapeutic target.

Inflammatory gastrointestinal (GI) diseases and malignancies are associated with growing morbidity and cancer-related mortality worldwide. GI tumor and inflammatory cells contain activated sphingolipid-metabolizing enzymes, including sphingosine kinase 1 (SphK1) and SphK2, that generate sphingosine-1-phosphate (S1P), a highly bioactive compound. Many inflammatory responses, including lymphocyte trafficking, are directed by circulatory S1P, present in high concentrations in both the plasma and the lymph of cancer patients. High fat and sugar diet, disbalanced intestinal flora, and obesity have recently been linked to activation of inflammation and SphK/S1P/S1P receptor (S1PR) signaling in various GI pathologies, including cancer. SphK1 overexpression and activation facilitate and enhance the development and progression of esophageal, gastric, and colon cancers. SphK/S1P axis, a mediator of inflammation in the tumor microenvironment, has recently been defined as a target for the treatment of GI disease states, including inflammatory bowel disease and colitis. Several SphK1 inhibitors and S1PR antagonists have been developed as novel anti-inflammatory and anticancer agents. In this review, we analyze the mechanisms of SphK/S1P signaling in GI tissues and critically appraise recent studies on the role of SphK/S1P/S1PR in inflammatory GI disorders and cancers. The potential role of SphK/S1PR inhibitors in the prevention and treatment of inflammation-mediated GI diseases, including GI cancer, is also evaluated.

HIF2α supports pro-metastatic behavior in pheochromocytomas/paragangliomas.

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix-receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.

Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR).

BACKGROUND/AIM: DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity. MATERIALS AND METHODS: Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR. RESULTS: By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept. CONCLUSION: All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases.

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.

Identification of CpG Sites of SERPINA5 Promoter with Opposite Methylation Patterns in Benign and Malignant Prostate Cells.

BACKGROUND/AIM: To date there has been no investigation into the epigenetic regulation of the serine protease inhibitor SERPINA5 in prostate cancer, where lack of this gene was considered to facilitate invasive growth patterns. MATERIALS AND METHODS: Methylation degrees of eight CpG sites of SERPINA5 were analyzed in normal and malignant prostate cells using nucleotide sequencing, methylation-specific high resolution melting and digital droplet PCR techniques. RESULTS: The methylation degree of five CpG sites significantly correlated with lower SERPINA5 expression levels. In contrast, two CpG sites (at -19 bp and -14 bp from the transcription start site) were hypermethylated in normal epithelial prostate cells, benign hyperplasic cells and low-invasive malignant LNCaP cells, whereas in aggressive DU-145 and PC-3 cell lines, these sites were essentially unmethylated. CONCLUSION: Novel methylation patterns of two distinct CpG sites of the SERPINA5 promoter may be useful for differentiating benign from malignant prostate disease.

Instructing Human Macrophage Polarization by Stiffness and Glycosaminoglycan Functionalization in 3D Collagen Networks.

Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.