Pathways regulating the expression of the immunomodulatory protein glycodelin in non­small cell lung cancer.

Glycodelin [gene name, progesterone associated endometrial protein (PAEP)] was initially described as an immune system modulator in reproduction. Today, it is also known to be expressed in several types of cancer, including non­small cell lung cancer (NSCLC). In this cancer type, the feasibility of its usage as a follow­up biomarker and its potential role as an immune system modulator were described. It is assumed that NSCLC tumours secrete glycodelin to overcome immune surveillance. Therefore, targeting glycodelin might be a future approach with which to weaken the immune system defence of NSCLC tumours. In this context, it is important to understand the regulatory pathways of PAEP/glycodelin expression, as these are mostly unknown so far. In this study, we analysed the influence of several inducers and of their downstream pathways on PAEP/glycodelin expression in a human lung adenocarcinoma carcinoma (ADC; H1975) and a human lung squamous cell carcinoma (SQCC) cell line (2106T). PAEP/glycodelin expression was notably stimulated by the canonical transforming growth factor (TGF)­ß pathway in SQCC cells and the PKC signalling cascade in both cell lines. The PI3K/AKT pathway inhibited PAEP/glycodelin expression in the ADC cells and an antagonizing role towards the other investigated signalling cascades is suggested herein. Furthermore, the mitogen­activated protein kinase kinase (MEK)/extracellular­signal regulated kinases (ERK) pathway was, to a lesser extent, found to be associated with increased PAEP/glycodelin amounts. The phosphoinositide 3­kinase (PI3K)/protein kinase B (AKT), MEK/ERK pathway and TGF­ß are targets of NSCLC drugs that are already approved or are currently under investigation. On the whole, the findings of this study provide evidence that inhibiting these targets affects the expression of glycodelin and its immunosuppressive effect in NSCLC tumours. Moreover, understanding the regulation of glycodelin expression may lead to the development of novel therapeutic approaches with which to weaken the immune system defence of NSCLC tumours in the future.

Development of New mRNA Markers for the Identification of Menstrual Blood.

BACKGROUND: The aim of this study was to screen 3 mRNA markers (i.e., PAEP, LAPR3, and HOXA10) with diverse expression in different body fluids and to develop a method for the identification of menstrual blood using these mRNA markers. METHODS: Body fluid (i.e., venous blood, menstrual blood, semen, and saliva) samples were collected and prepared under differing environmental conditions (temperature, humidity and time), and RNA was extracted and reverse transcribed. The expression specificity of these markers was assessed using TaqMan probe qPCR. RESULTS: A high mean cycle threshold value corresponds to a lower expression level. The mean cycle threshold value of the LAPR3, HOXA10, and PAEP genes are 8.37, 8.73, 4.67 in menstrual blood respectively. LAPR3 and PAEP were only expressed in menstrual blood. HOXA10 were expressed in blood, menstrual blood, and semen. No significant differences were found while the mean cycle threshold of MMP11 and PAEP were compared in the menstrual blood under common environmental conditions. There were no observed differences in the expression of the target genes in women of different ages and at different menstrual phases. The sensitivity of the expression of the 3 target genes could be examined in fluid amount range from 1 to 32 µl of body fluid. The expression of PAEP differed markedly from the expression of LAPR3 and HOXA10 in menstrual blood stains tested using mRNA-based assays (p<0.001). CONCLUSIONS: These markers, particularly PAEP, can likely be used for the identification of menstrual blood in certain forensic cases.

Dysregulation of GdA Expression in Endometrium of Women With Endometriosis: Implication for Endometrial Receptivity.

Glycodelin-A (GdA) has been proposed to represent a potential biomarker of endometrial function, but little is known about its expression during the different phases of the menstrual cycle and under pathological conditions. In the light of its potential importance also in embryo implantation, we aimed to evaluate the expression profile of GdA as well as the presence of different glycosylated glycoforms and the immunolocalization in endometrial tissue from women with endometriosis and in women with proven fertility, at different times during the menstrual cycle. Our results showed that GdA is synthesized by endometrial epithelial and stromal cells, both in healthy endometrium and eutopic endometrium from women with endometriosis, with a profile including several glycosylated glycoforms, differentially expressed in each phase of the menstrual cycle. During the secretory phase, a significant increase in GdA protein expression, with a different glycoforms profile, was observed in endometriotic eutopic endometrium. Protein localization in eutopic endometrial tissue resulted significantly different in comparison with endometrium from women with proven fertility. This study indicate that GdA is a complex glycoprotein including up to 6 different glycoforms specifically expressed during the different phase of the menstrual cycle; in pathologic conditions such as endometriosis, the expression profile is altered possibly related to the impaired endometrial receptivity.

Downregulation of genes related to immune and inflammatory response in IVF implantation failure cases under controlled ovarian stimulation.

PROBLEM: Implantation failure (IF) even after the good-quality embryo transfer (ET) is main obstacle in in vitro fertilization (IVF). We aim to study the genomics of endometrial receptivity in IF patients under controlled ovarian stimulation (COS) during which ET is generally practised in IVF. METHOD OF STUDY: Endometrial gene expression profiling in IF patients (n=10) and oocyte donors (n=8) were compared during window of implantation under COS by microarray. Enrichment analysis of microarray data was performed to determine dysregulated pathways. Microarray results were validated by real-time PCR. Localization of genes related to immune response (progestagen-associated endometrial protein (PAEP), leukaemia inhibitory factor (LIF), interleukin-6 signal transducer (IL6ST) was detected by immunohistochemistry. RESULTS: The gene ontology, pathway analysis and enrichment mapping revealed significant downregulation in activation and regulation of immune and inflammation response in IF patients under COS. The lower expression of PAEP, LIF and IL6ST in cases compared to controls by real time and immunohistochemistry suggests the functional importance of these genes. CONCLUSION: Importance of immune and inflammatory response in endometrial receptivity adds on to the current knowledge of gene expression profile in IF under COS. The panel of genes involved in these pathways would be useful in determining further line of treatment for IF during IVF.