Since recent studies revealed the feasibility to detect blood-based microRNAs (miRNAs, miRs) in breast cancer (BC) patients a new field has been opened for circulating miRNAs as potential biomarkers in BC. In this pilot study, we evaluated to our knowledge for the first time whether distinct pattern of urinary miRNAs might be also applicable as innovative biomarkers for BC detection. Urinary miRNA expression levels of nine BC-related miRNAs (miR-21, miR-34a, miR-125b, miR-155, miR-195, miR-200b, miR-200c, miR-375, miR-451) from 24 untreated, primary BC patients and 24 healthy controls were quantified by realtime-PCR. The receiver operating characteristic analyses (ROC) and logistic regression were calculated to assess discriminatory accuracy. Significant differences were found in the expression of four BC-associated miRNAs quantified as median miRNA expression levels. Urinary miR-155 levels were significantly higher in BC patients compared to healthy controls (1.49vs.0.25; p < 0.001). In contrast, compared to healthy controls, BC patients exhibited significantly lower urinary expression levels of miR-21 (2.27vs.5.07; p < 0.001), miR-125b (0.71vs.1.62; p < 0.001), and miR-451 (0.02vs.0.59 p = 0.004), respectively. The ROC including all miRNAs as well as the group of the four significant deregulated miRNAs separated BC patients from healthy controls with a very high (area under the receiver operating characteristic curve [AUC] = 0.932) and high accuracy (AUC = 0.887), respectively. We were able to demonstrate for the first time the feasibility to detect distinct BC-dependent urinary miRNA profiles. The expression levels of four urinary miRNAs were specifically altered in our cohort of BC patients compared to healthy controls. This distinct pattern offers the possibility for a specific discrimination between healthy women and primary BC patients. This sustains the potential role of urinary miRNAs as non-invasive innovative urine-based biomarkers for BC detection.

Background: Cancer–testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors.Methods: The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival.Results: Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively).Conclusions: Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.

Estrogen receptor alpha (ERa/ESR1) expression is regulated by alternative splicing. Its most frequently detectable exon7 skipping isoform (ERaD7) is a dominant negative variant. Elevated expression of ERaD7 was already detected in endometrial cancer (EC), while its potential prognostic significance has not been characterized so far. Exon7 contains potential binding sites for the two functional splicing regulatory opponents, HNRNPG and HTRA2-BETA1 known to trigger opposite effects on EC outcome. This study served to elucidate the influence of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing regulation and the impact of ERaD7 concentration on type 1 EC outcome. Functional in vitro experiments for HNRNPG and HTRA2-BETA1 in regard to the regulatory impact on endogenous and exogenous ERaD7 splicing were performed. Additionally, real-time PCR determined mRNA levels of ERaD7, HNRNPG and HTRA2-BETA1 in 116 type 1 EC patients. HNRNPG and HTRA2-BETA1 were found to be specific regulators of ERa exon7 splicing. While HTRA2-BETA1 promoted exon7 inclusion, HNRNPG antagonized this effect by inducing exon7 skipping (p = 0.004). ERaD7 was detected in 71 out of 116 type 1 EC specimens. Statistical analyses revealed an inverse correlation between ERaD7 mRNA levels and tumor grading (p = 0.029), FIGO stage (p = 0.033) as well as lymph node metastases (p = 0.032), respectively. Furthermore, higher ERaD7 expression could be correlated to an improved disease-specific survival (p = 0.034). Our study demonstrates antagonistic regulatory effects of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing with potential impact on type 1 EC clinical outcome due to the consecutively variable expression levels of the ERa isoform D7.

Ovarian carcinoma is a gynecological cancer with poor long-term survival rates when detected at advanced disease stages. Early symptoms are non-specific, and currently, there are no adequate strategies to identify this disease at an early stage when much higher survival rates can be expected. Ovarian carcinoma is a heterogeneous disease, with various histotypes originating from different cells and tissues, and is characterized by distinct somatic mutations, progression profiles, and treatment responses. Our study presents a targeted metabolomics approach, characterizing seven different ovarian (cancer-) cell lines according to their extracellular, intracellular, and RNA-derived modified nucleoside profiles. Moreover, these data were correlated with transcriptomics data to elucidate the underlying mechanisms. Modified nucleosides are excreted in higher amounts in cancer cell lines due to their altered DNA/RNA metabolism. This study shows that seven different ovarian cancer cell lines, representing different molecular subtypes, can be discriminated according to their specific nucleoside pattern. We suggest modified nucleosides as strong biomarker candidates for ovarian cancer with the potential for subtype-specific discrimination. Extracellular modified nucleosides have the highest potential in the distinguishing of cell lines between control cell lines and themselves, and represent the closest to a desirable, non-invasive biomarker, since they accumulate in blood and urine.

Endometrial carcinoma (EC) is the most common malignancy of the female reproductive tract, with increasing incidence driven by aging populations and obesity. While molecular classification has improved diagnostic precision, the identification of clinically relevant metabolic biomarkers remains incomplete, and targeted therapies are not yet standardized. In this study, we investigated metabolic alterations in four EC cell lines (AN3-CA, EFE-184, HEC-1B and MFE-296) compared to non-malignant controls under normoxic and stress conditions (hypoxia and lactic acidosis) to identify metabolomic differences with potential clinical relevance. Untargeted gas chromatography–mass spectrometry (GC/MS) and targeted liquid chromatography–mass spectrometry (LC/MS) profiling revealed two distinct metabolic subtypes of EC. Cells of metabolic subtype 1 (AN3-CA and EFE-184) exhibited high biosynthetic and energy demands, enhanced cholesterol and hexosyl-ceramides synthesis and increased RNA stability, consistent with classical cancer-associated metabolic reprogramming. Cells of metabolic subtype 2 (HEC-1B and MFE-296) displayed a phospholipid-dominant metabolic profile and greater hypoxia tolerance, suggesting enhanced tumor aggressiveness and metastatic potential. Key metabolic findings were validated via real-time quantitative PCR. This study identifies and characterizes distinct metabolic subtypes of EC within the investigated cancer cell lines, thereby contributing to a better understanding of tumor heterogeneity. The results provide a basis for potential diagnostic differentiation based on specific metabolic profiles and may support the identification of novel therapeutic targets. Further validation in three-dimensional culture models and ultimately patient-derived samples is required to assess clinical relevance and integration with current molecular classifications.

Background: Breast cancer is the most common type of cancer worldwide. Cyclin-dependent kinase inhibition is one of the backbones of metastatic breast cancer therapy. However, there are a significant number of therapy failures. This study evaluates the biomarker potential of microRNAs for the prediction of a therapy response under cyclin-dependent kinase inhibition. Methods: This study comprises the analysis of intracellular and extracellular microRNA-expression-level alterations of 56 microRNAs under palbociclib mono as well as combination therapy with letrozole. Breast cancer cell lines BT-474, MCF-7 and HS-578T were analyzed using qPCR. Results: A palbociclib-induced microRNA signature could be detected intracellularly as well as extracellularly. Intracellular miR-10a, miR-15b, miR-21, miR-23a and miR-23c were constantly regulated in all three cell lines, whereas let-7b, let-7d, miR-15a, miR-17, miR-18a, miR-20a, miR-191 and miR301a_3p were regulated only in hormone-receptor-positive cells. Extracellular miR-100, miR-10b and miR-182 were constantly regulated across all cell lines, whereas miR-17 was regulated only in hormone-receptor-positive cells. Conclusions: Because they are secreted and significantly upregulated in the microenvironment of tumor cells, miRs-100, -10b and -182 are promising circulating biomarkers that can be used to predict or detect therapy responses under CDK inhibition. MiR-10a, miR-15b, miR-21, miR-23a and miR-23c are potential tissue-based biomarkers.