Accumulation of GAS5 in exosomes is a marker of apoptosis induction

Long non-coding RNAs (lncRNAs) are key regulatory molecules in many fundamental cellular processes and their deregulation is assumed to contribute to carcinogenesis. Exosomal lncRNAs are thought to be involved in the dissemination of cell signals to control local cellular microenvironments. In the current study, exosomal expression of growth arrest specific 5 (GAS5), an inhibitor of cell proliferation and promoter of apoptosis, was evaluated in apoptotic processes initiated by different mechanisms. Therefore, MCF-7 and MDA-MB-231 breast cancer cells were treated with Taxol (2 and 10 nM) and bleomycin (2 and 10 ng/ml) for 24 h. Following cell viability determination and measurement of apoptosis, cellular and exosomal expression levels of GAS5 were investigated using a quantitative polymerase chain reaction assay. The findings indicate that Taxol is more toxic than bleomycin at the indicated doses and the effect was more evident in the MCF-7 cells. Despite varying toxicity rates, comparable levels of apoptotic nucleosomes were measured between Taxol-and bleomycin-treated cells. Upon drug treatment, cellular expression levels of GAS rose (<= 1.5-fold) in the two cell lines. It appears that even a small increase in cellular expression leads to exosomal enrichment, as the accumulation of GAS5 in exosomes was marked in the MCF-7 cells (<= 5.8-fold). Compared with the MCF-7 cells, the extent of GAS5 enrichment in the exosomes secreted from MDA-MB-231 cells was moderate (<= 1.9-fold), potentially as a result of reduced cell death. The present study indicates that GAS5 accumulation in exosomes is a prevalent event in apoptotic processes that are initiated by different mechanisms

Potential of circulating nucleosome-associated histone modifications in cancer

Posttranslational modifications of histone proteins (PTHMs) are involved in the regulation of chromatin packaging and gene expression thus affecting cellular homeostasis. Enzymatic machinery that set PTHMs is often deregulated in cancer resulting in altered global patterns of PTHMs in various cancer types. Detection of cancer-related histone marks in biological fluids such as plasma and serum could serve as useful biomarkers in cancer detection, diagnosis and management. Such biomarkers offer many advantages, such as being minimally invasive and easily accessible. Reflecting this, increasing amounts of data on the impact of circulating histone marks are being published. Here we provide a short insight into PTHMs and their potential utility as circulating cancer biomarkers

The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients

Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests. Furthermore, serial monitoring of ctDNA over the course of systemic and targeted therapies not only allows unparalleled efficacy assessments but also enables the identification of patients who are at risk of progression or recurrence. Droplet digital PCR (ddPCR) is a powerful technique for the detection and monitoring of ctDNA. Due to its relatively high accuracy, sensitivity, reproducibility, and capacity for absolute quantification, it is increasingly used as a tool for managing cancer patients through liquid biopsies. In this review paper, we gauge the clinical utility of ddPCR as a technique for mutational profiling in breast cancer patients and focus on HER2, PIK3CA, ESR1, and TP53, which represent the most frequently mutated genes in breast cancers

Global quantification of heterochromatin-associated histone methylations in cell lines with differential sensitivity to ionizing radiation

Histone modifications are involved in the DNA damage response (DDR). Here, by utilizing an ELISA immunoassay we assessed the methylation at H3K9 (H3K9me2 and H3K9me3) in two cell lines with differential sensitivity to radiation-induced apoptosis, He La (sensitive) and MCF-7 (resistant). We found that DNA damage induction by gamma-irradiation leads to considerable accumulation (up to 5-fold) of H3K9me2 and H3K9me3, but not of H4K20me3 (control modification) in MCF-7 cells (p<0.05). Interestingly, a lower dose (2 Gy) was more effective than 5 Gy. In He La cells a smaller effect (approx. 1.5-1.8-fold) was evident only at 5 Gy. In conclusion, our findings reveal that DNA damage leads to specific accumulation of H3K9me2 and H3K9me3 in a cell-type specific manner

PCA3 Silencing Sensitizes Prostate Cancer Cells to Enzalutamide-mediated Androgen Receptor Blockade

Background/Aim: Prostate cancer (PCa) is an androgen-dependent disease. Novel anti-androgens (i.e. enzalutamide) have recently been developed for the treatment of patients with metastatic castration-resistant prostate cancer (CRPC). Evidence is accumulating that prostate cancer antigen 3 (PCA3) is involved in androgen receptor (AR) signaling. Here, in combination with enzalutamide-mediated AR blockade, we investigated the effect of PCA3 targeting on the viability of PCa cells. Materials and Methods: In hormone-sensitive LNCaP cells, AR-overexpressing LNCaP-AR(+) cells and VCaP cells (representing CRPC), PCA3 was silenced using siRNA oligonucleotides. Gene expression and cell viability was assessed in PCA3-silenced and/or AR-blocked cells. Results: PCA3 targeting reduced the expression of AR-related genes (i.e. prostate-specific antigen (PSA) and prostate-specific transcript 1 (non-protein coding) (PCGEM1)) and potentiated the effect of enzalutamide. Proliferation of PCa cells was suppressed upon PCA3 silencing with a greater effect in LNCaP-AR(+) cells. Furthermore, PCA3 silencing sensitized PCa cells to enzalutamide-induced loss of cell growth. Conclusion: PCA3, as a therapeutic target in PCa, might be used to potentiate AR antagonists

Circulating lncRNA H19 may be a useful marker of response to neoadjuvant chemotherapy in breast cancer

BACKGROUND: Novel biomarkers are needed to predict the effectiveness of the treatment of presurgical neoadjuvant chemotherapy (NAC) in breast cancer (BC)

Androgen Stimulation of PCA3 and miR-141 and Their Release from Prostate Cancer Cells

Objective: Prostate cancer antigen 3 (PCA3) and microRNA-141 (miR-141) are emerging molecules in prostate cancer (PCa) pathogenesis and have been shown to be involved in androgen signaling. In this original research, we designed an experimental cell model with androgen-sensitive LNCaP cells to comparatively assess the extent of androgen responsiveness of PCA3-mRNA and miR-141 along with prostate specific antigen (PSA)- mRNA and their release into culture medium. These molecules were also measured in the plasma of the patients with early PCa which is considered to be analogous to androgenresponsive cells. Materials and Methods: In this experimental study, LNCaP cells were exposed to androgen ablation for 48 hours and treated then with dihydrotestosterone (DHT) for 24 hours. Expression of all three RNA molecules in cells, culture medium or plasma was measured by quantitative polymerase chain reaction (qPCR). Results: Our results show that DHT differentially affects the expression of these molecules. PCA3 was the most evidently induced molecule (up to 400-fold, p0.05). With regard to the release into culture medium, a similar picture was observed except for PCA3. PCA3 was below the detection level despite its high stimulation. DHT treatment led to a significant release of PSA-mRNA (up to 12-fold). Similar to its induction pattern in LNCaP cells, miR-141 was released at a limited quantity into the medium (up to 1.7- fold, p=0.07). In plasma, only PCA3 differed significantly between the patients and healthy subjects (p=0.001). Conclusion: Our findings indicate that PCa-related RNA molecules respond differentially to androgen stimulation suggesting differential regulation by androgens

Relevance of Histone Marks H3K9me3 and H4K20me3 in Cancer

Background: Circulating nucleosomes are valuable biomarkers for therapy monitoring and estimation of prognosis in cancer disease. While epigenetic and genetic modifications of DNA have been reported in blood of cancer patients, little is known about modifications of histones on circulating nucleosomes. Patients and Methods: Sera of 45 cancer patients (21 colorectal, 4 pancreatic, 15 breast, 5 lung cancer), 12 patients with benign gastrointestinal and inflammatory diseases, and 28 healthy individuals were investigated. Histone modifications were detected by chromatin-immunoprecipitation (ChIP) using antibodies for triple hi stone methylations at sites wH3K9me3 and H4K20me3 and subsequent real-time polymerase chain reaction using primers for the centromeric satellites SAT2. Additionally, the amount of circulating nucleosomes, as well as of carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured. Results: Levels of SAT2 on H3K9me3 (median 0.507 ng/ml) and on H4K20me3 (0.292 ng/ml) were elevated in sera of patients with breast cancer when compared with healthy controls (0.049 and 0.035 ng/ml), but were lower in patients with colorectal cancer (0.039 and 0.027 ng/ml). Both histone marks were correlated with each other but did not correlate with CEA or CA 19-9 in cancer patients. When H3K9me3 and H4K20me3 were normalized to nucleosome content in sera, ratios were significantly higher in all types of cancer as well as in colorectal and breast subtypes when compared with healthy controls. Best discrimination was achieved by normalized H4K20me3 reaching areas under the curves (AUC) of 79.1%, 90.4% and 81.2% in receiver operating characteristic (ROC) curves of these three comparisons. Conclusion: SAT2 levels on H3K9me3 and H4K20me3 are up-regulated in breast cancer and down-regulated in colorectal cancer. Normalization to total nucleosome content enables better discrimination between cancer and control groups

Analysis of circulating microRNAs during adjuvant chemotherapy in patients with luminal A breast cancer

Breast cancer is a complex disease displaying different profiles involving genetic as well as epigenetic factors. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. Recent studies demonstrated that miRNAs may display great potential for the development of novel biomarkers and therapeutic targets. In the present study, the levels of miR-21 and miR-145 were analyzed in the peripheral blood of 52 patients with luminal A breast cancer. miRNA expression was determined in serum samples from matched pre- and post-treatment patients with breast cancer by quantitative polymerase chain reaction. There were no statistically significant differences in miR-145 and miR-21 levels between pre- and post-treatment samples. In addition, the miRNA levels were not found to be associated with the clinical parameters