Evaluation of circulating microRNAs as non-invasive biomarkers in the diagnosis of ovarian cancer: a case-control study.

PURPOSE: Ovarian cancer is the seventh most frequent form of malignant diseases in women worldwide and over 150,000 women die from it every year. More than 70 percent of all ovarian cancer patients are diagnosed at a late-stage disease with poor prognosis necessitating the development of sufficient screening biomarkers. MicroRNAs displayed promising potential as early diagnostics in various malignant diseases including ovarian cancer. The presented study aimed at identifying single microRNAs and microRNA combinations detecting ovarian cancer in vitro and in vivo. METHODS: Intracellular, extracellular and urinary microRNA expression levels of twelve microRNAs (let-7a, let-7d, miR-10a, miR-15a, miR-15b, miR-19b, miR-20a, miR-21, miR-100, miR-125b, miR-155, miR-222) were quantified performing quantitative real-time-PCR. Therefore, the three ovarian cancer cell lines SK-OV-3, OAW-42, EFO-27 as well as urine samples of ovarian cancer patients and healthy controls were analyzed. RESULTS: MiR-15a, miR-20a and miR-222 showed expression level alterations extracellularly, whereas miR-125b did intracellularly across the analyzed cell lines. MicroRNA expression alterations in single cell lines suggest subtype specificity in both compartments. Hypoxia and acidosis showed scarce effects on single miRNA expression levels only. Furthermore, we were able to demonstrate the feasibility to clearly detect the 12 miRNAs in urine samples. In urine, miR-15a was upregulated whereas let-7a was down-regulated in ovarian cancer patients. CONCLUSION: Intracellular, extracellular and urinary microRNA expression alterations emphasize their great potential as biomarkers in liquid biopsies. Especially, miR-15a and let-7a qualify for possible circulating biomarkers in liquid biopsies of ovarian cancer patients.

Mutually distinguishing microRNA signatures of breast, ovarian and endometrial cancers in vitro.

Early diagnosis and therapy in the first stages of a malignant disease is the most crucial factor for successful cancer treatment and recovery. Currently, there is a high demand for novel diagnostic tools that indicate neoplasms in the first or pre­malignant stages. MicroRNAs (miRNA or miR) are small non­coding RNAs that may act as oncogenes and downregulate tumor­suppressor genes. The detection and mutual discrimination of the three common female malignant neoplasia types breast (BC), ovarian (OC) and endometrial cancer (EC) could be enabled by identification of tumor entity­specific miRNA expression differences. In the present study, the relative expression levels of 25 BC, EC and OC­related miRNAs were assessed by reverse transcription­quantitative PCR and determined using the 2­ΔΔCq method for normalization against the mean of four housekeeping genes. Expression levels of all miRNAs were analyzed by regression against cell line as a factor. An expression level­based discrimination between BC and OC cell types was obtained for a subgroup of ten different miRNA types. miR­30 family genes, as well as three other miRNAs, were found to be uniformly upregulated in OC cells compared with BC cells. BC and EC cells could be distinguished by the expression profiles of six specific miRNAs. In addition, four miRNAs were differentially expressed between EC and OC cells. In conclusion, miRNAs were identified as a potential novel tool to detect and mutually discriminate between BC, OC and EC. Based on a subset of 25 clinically relevant human miRNA types, the present study could significantly discriminate between these three female cancer types by means of their expression levels. For further verification and validation of miRNA­based biomarker expression signatures that enable valuable tumor detection and characterization in routine screening or potential therapy monitoring, additional and extended in vitro analyses, followed by translational studies utilizing patients' tissue and liquid biopsy materials, are required.

Endoxifen and fulvestrant regulate estrogen-receptor α and related DEADbox proteins.

Breast cancer (BC) represents the most common type of cancer in females worldwide. Endocrine therapy evolved as one of the main concepts in treatment of hormone-receptor positive BC. Current research focuses on the elucidation of tumour resistance mechanisms against endocrine therapy. In a translational in vitro approach, potential regulatory effects of clinically implemented BC anti-oestrogens on ERα, its coactivators DDX5, DDX17 and other DEADbox proteins as well as on the proliferation markers cyclin D1 and Ki67 were investigated on both the RNA and protein level. BC in vitro models for hormone-receptor positive (MCF-7, T-47D) and hormone-receptor negative cells (BT-20) were subjected to endocrine therapy. Anti-oestrogen-dependent expression regulation of target genes on the transcriptional and translational level was quantified and statistically assessed. Endocrine therapy decreases the expression levels of Ki67, cyclin D1 and ERα in hormone-receptor positive cells. In the hormone-receptor negative cells, the three parameters remained stable after endocrine therapy. Endoxifen triggers a downregulation of DDX5 and DDX23 in MCF-7 cells. Fulvestrant treatment downregulates the expression levels of all investigated DEADbox proteins in MCF-7 cells. In T-47D cells, endoxifen and fulvestrant lead to a decrease of all target gene expression levels. Interestingly, endocrine therapy affects DEADbox RNA expression levels in BT-20 cells, too. However, this result could only be confirmed for DDX1, immunocytologically. The investigated DEADbox proteins appear to correlate with the oestrogen-dependent tumourigenesis in hormone-receptor positive BC and show expression alterations after endocrine treatment.

Circulating non­coding RNA­biomarker potential in neoadjuvant chemotherapy of triple negative breast cancer?

Due to the positive association between neoadjuvant chemotherapy (NACT) and the promising early response rates of patients with triple negative breast cancer (TNBC), including probabilities of pathological complete response, NACT is increasingly used in TNBC management. Liquid biopsy­based biomarkers with the power to diagnose the early response to NACT may support established monitoring tools, which are to a certain extent imprecise and costly. Simple serum­ or urine­based analyses of non­coding RNA (ncRNA) expression may allow for fast, minimally­invasive testing and timely adjustment of the therapy regimen. The present study investigated breast cancer­related ncRNAs [microRNA (miR)­7, ­9, ­15a, ­17, ­18a, ­19b, ­21, ­30b, ­222 and ­320c, PIWI­interacting RNA­36743 and GlyCCC2] in triple positive BT­474 cells and three TNBC cell lines (BT­20, HS­578T and MDA­MB­231) treated with various chemotherapeutic agents using reverse transcription­quantitative PCR. Intracellular and secreted microvesicular ncRNA expression levels were analysed using a multivariable statistical regression analysis. Chemotherapy­driven effects were investigated by analysing cell cycle determinants at the mRNA and protein levels. Serum and urine specimens from 8 patients with TNBC were compared with 10 healthy females using two­sample t­tests. Samples from the patients with TNBC were compared at two time points. Chemotherapeutic treatments induced distinct changes in ncRNA expression in TNBC cell lines and the BT­474 cell line in intra­ and extracellular compartments. Serum and urine­based ncRNA expression analysis was able to discriminate between patients with TNBC and controls. Time point comparisons in the urine samples of patients with TNBC revealed a general rise in the level of ncRNA. Serum data suggested a potential association between piR­36743, miR­17, ­19b and ­30b expression levels and an NACT­driven complete clinical response. The present study highlighted the potential of ncRNAs as liquid biopsy­based biomarkers in TNBC chemotherapy treatment. The ncRNAs tested in the present study have been previously investigated for their involvement in BC or TNBC chemotherapy responses; however, these previous studies were restricted to patient tissue or in vitro models. The data from the present study offer novel insight into ncRNA expression in liquid samples from patients with TNBC, and the study serves as an initial step in the evaluation of ncRNAs as diagnostic biomarkers in the monitoring of TNBC therapy.

Alternative splicing of synuclein gamma in endometrial cancer: identification of a novel isoform.

Synuclein gamma (SNCG) is under consideration as a potential biomarker in cancer biology. Up to date four different SNCG variants are described. Due to growing evidence suggesting correlations between aberrant alternative splicing processes and cancer progression, we investigated the effects of peritumoural conditions on expression pattern of SNCG in endometrial cancer (EC) in vitro. Compared to breast cancer cell lines, mRNA expression levels of all known SNCG isoforms 1-4 are significantly reduced in EC cell lines. We identified a novel alternatively spliced variant of isoform 2 (isoform 2 short) which is found highly expressed in EC cell lines. Hypoxia and acidosis trigger an up-regulation of isoform 2 short. EC cell lines are characterized by low SNCG protein levels under control conditions, but exhibit a significant increase triggered by hypoxia and acidosis. In addition we analysed the potential association between SNCG protein expression and clinico-pathological parameters in human EC samples. Our findings indicate a grade-dependent induction of SNCG protein expression in endometrial cancer. We identified for the first time a novel isoform of SNCG that is found specifically expressed in EC. Our results also strongly indicate the existence of a corresponding protein of isoform 2 short that potentially plays a critical role in EC cancer progression.